TW202134277A - N-terminal scfv multispecific binding molecules - Google Patents

Abstract

Multispecific binding molecules (MBMs) comprising an N-terminal scFv, a first Fab and a second Fab, MBM conjugates comprising the MBMs and cytotoxic or cytostatic agents, pharmaceutical compositions ontaining the MBMs and MBM conjugates, methods of using the MBMs, MBM conjugates and pharmaceutical compositions for treating cancer, nucleic acids encoding the MBMs, cells engineered to express the MBMs, and methods of producing MBMs.

Description

N-terminal scFv multispecific binding molecule

Cross-reference of related applications

This application is a claim for the priority of U.S. Provisional Application No. 62/930,916 filed on November 2019, the content of which is incorporated herein by reference in its entirety. Sequence Listing

This application contains a sequence listing electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy made on November 2, 2020 is named RGN-003TW_SL.txt and is 60,900 bytes in size.

Most naturally occurring antibody molecules generally include two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each heavy chain and light chain polypeptide system contains a variable domain (variable region) (generally the amino terminal part of the polypeptide chain), and the variable domain system includes a binding region capable of interacting with an antigen. Each heavy chain and light chain polypeptide system includes a constant domain (generally a carboxy-terminal part).

In the past 20 years, recombinant monoclonal antibodies produced by single clones of cells or cell lines have emerged as a very successful class of biopharmaceuticals for the treatment of various diseases. Antibody-based therapeutics have been successfully used to treat various diseases, including cancer and autoimmune/inflammatory disorders.

Due to the biological complexity of certain diseases, antibodies that target more than one antigen or epitope may be more effective than single antibodies in treating specific symptoms. See, for example, Lindzen et al. , 2010, Proc. Natl. Acad. Sci. 107(28): 12550-12563; Nagorsen and Baeuerle, 2011, Exp. Cell Res. 317(9): 1255-60. These antibodies provide a guarantee for better treatment control. For example, there is a need to improve target specificity in order to reduce the off-target effects associated with many antibody therapies, especially antibody-based immunotherapies. In addition, multispecific antibodies provide novel therapeutic strategies, such as coordinated targeting of multiple cellular receptors, especially in the context of immunotherapy.

This article provides a multispecific binding molecule ("MBM"), and the MBM contains at least three antigen-binding sites ("ABS"), two of which are Fabs and the third is scFv, the scFv is connected to one of the Fabs at the N-terminus of the VH domain. The MBM herein contains an Fc domain, which is composed of two heavy chain Fc domains that bind to each other. Each polypeptide chain including an Fc domain and any related polypeptide chains are referred to as "half antibodies" in the text.

The exemplary MBM system in this article is shown in Figure 1, and its variant system is shown in Figures 2 and 3.

The typical MBM system herein includes two half antibodies connected via its Fc domain. The half-antibody shown on the left side of Figures 1 to 3, from N- to C-terminal, includes: • scFv, which is an optional presence of VH(1) and VL(3) (in any order) The linker (2) is connected; • The linker (4) that exists as needed; • The first Fab ("Fab1"), which is composed of the following: o VH(5) and constant domain (6), It is the CH1 domain in the example of Fig. 1, but it can be different types of constant domains as shown in Fig. 2A and Fig. 2C, such as CL or CH3, which helps Fab heterodimerization, connected to the following o VL(10) And the constant domain (11), which is the CL domain in the example in Figure 1, but can be different types of constant domains, such as CH1 or CH3, as shown in Figure 2A and Figure 3C; • Hinge domain ( 7); • Fc domain, which is composed of CH2 domain (8) and CH3 domain (9), as shown in Figure 3, may contain one or more mutations (such as star mutations or club-and-socket structures) (knob-in-hole mutation) to help heterodimerization.

The semi-antibody system on the left is composed of two polypeptide chains. The first polypeptide chain system includes VL (10) and constant domain (11) and is connected to the second polypeptide chain including the remaining regions as shown in Figures 1 to 3 .

The half-antibody shown on the right side of Figure 1, from N- to C-terminal, includes: • The second Fab ("Fab2"), which is composed of: o VH (12) and constant domain (13), It is the CH1 domain in the example of Fig. 1, but it can be different types of constant domains as shown in Fig. 2B and Fig. 2D, such as CL or CH3, which helps Fab heterodimerization, and o VL(17) and constant domains. (18) Connected, which is the CL domain in the example of Figure 1, but can be different types of constant domains as shown in Figure 2B and Figure 2D, such as CH1 or CH3; • Hinge domains (14) that exist as needed ); • The Fc domain, which is composed of a CH2 domain (15) and a CH3 domain (16), as shown in Figure 3, can contain one or more mutations (such as star-shaped mutations or club-and-hole mutations) for Help the purification and/or combination of heterodimers.

The right semi-antibody system is composed of two polypeptide chains. The first polypeptide chain system includes VL (17) and constant domain (18) and is connected to the second polypeptide chain including the remaining regions as shown in Figures 1 to 3 .

The complete MBM is formed by linking two half-antibodies to form an Fc region through two Fc domains. The MBM has a scFv with a first antigen-binding site ("ABS1") and a second antigen-binding site ("ABS2") Fab1 and Fab3 with the third antigen binding site ("ABS3").

The variants of the MBM herein shown in Figures 1 to 3 are not intended to be limiting; wherein the MBM herein can include any combination of the modifications described in Figures 1 to 3 and section 6.2 below. Furthermore, the first or second polypeptide chain or the left or right half antibody is only for convenience and is not intended to convey that these polypeptide chains or half antibody systems are manufactured or assembled in any particular order.

The ABS1, ABS2, and ABS3 of MBM herein are each bound to target molecules, such as antigens on the surface of cells. Preferably, the scFv, Fab1, and Fab2 of MBM are selected so that each of ABS1, ABS2, and ABS3 can be combined with their respective targets at the same time. In some specific examples, each of ABS1, ABS2, and ABS3 of MBM can bind to different target molecules, or alternatively, two of ABS1, ABS2, and ABS3 can bind to different regions on the same target molecule.

Advantageously, MBM that binds at least two or more different target molecules can be used, for example, to preferentially target specific tissue types that exhibit these two or more target molecules.

The scFvs that can be used in the MBM herein are described in section 6.2.1 and specific specific examples 1 to 12, 19 to 20, 31 to 35, and 55 to 60 below. The Fab lines of MBM that can be used herein are described in section 6.2.2 and specific specific examples 1-12, 19-20, 22-23, 28-30, and 36-54 below. The scFv can be connected to Fab1 via a peptide linker, for example. The linker system that can be used to connect scFv and Fab1 is described in section 6.2.3 and specific examples 13 to 18 below. The Fc domains of MBM that can be used herein are described in section 6.2.4 and specific specific examples 24 to 26 below.

This article further provides drug conjugates including the MBM herein (for convenience, this article is referred to as "antibody-drug conjugate" or "ADC (antibody-drug conjugates)"). The features of the exemplary ADC are described in section 6.3 and specific specific example 61 below.

This article further provides nucleic acids encoding the MBM herein. The nucleic acid encoding the MBM may be a single nucleic acid (for example, a vector encoding the polypeptide chains of all MBM) or a plurality of nucleic acids (for example, two or more vectors encoding the polypeptide chains of different MBM). This article further provides host cells and cell lines engineered to express the nucleic acid and the MBM herein. This article further provides methods for manufacturing the MBM of this article. Exemplary nucleic acids, host cells, cell lines, and methods of making MBM are described in section 6.5 and specific specific examples 78 to 83 below.

This article further provides a pharmaceutical composition including the MBM and ADC described herein. Exemplary pharmaceutical compositions are described in Section 6.6 and Specific Specific Examples 62 below.

The article further provides methods for using the MBM, ADC, and pharmaceutical composition herein, for example, for the treatment of cancer. The exemplary method is described in section 6.7 and specific specific examples 63 to 77 below.

6.1 definition

As used in the text, the following terms are expected to have the following meanings:

Antigen binding site or ABS : The term "antigen binding site" or "ABS" as used herein refers to the portion of the MBM that can specifically, non-covalently and reversibly bind to the target molecule. The MBM in this article includes the first ABS ("ABS1") which is the scFv part, the ABS which is the Fab part ("ABS2") and the third ABS which is the Fab part ("ABS3").

Binding : The term "binding" in the case of MBM refers to a functional relationship between two or more polypeptide chains. In particular, the term "binding" refers to two or more polypeptides, for example, non-covalently through intermolecular interaction or covalently through one or more disulfide bridges or chemical cross-links to bind to each other, so as to produce functionality MBM, where ABS1, ABS2 and ABS3 can bind to its individual targets. Examples of binding that may exist in MBM herein include (but are not limited to) binding between homodimer or heterodimer Fc domains in the Fc region, binding between VH and VL domains in Fab or scFv, Fab The binding between CH1 and CL, the binding between CH3 and CH3 in the substituted Fab.

Complementarity determining region or CDR : The term "complementarity determining region" or "CDR" as used herein refers to an amino acid sequence that confers antigen specificity and binding affinity within the variable region of an antibody. Generally speaking, there are three CDRs (CDR-H1, CDR-H2, HCDR-H3) in each heavy chain variable region and three CDRs (CDR1-L1, CDR-L2, CDR-H3) in each light chain variable region. -L3). Exemplary definition methods that can be used to identify CDR boundaries include, for example, Kabat definition, Chothia definition, ABS definition, and IMGT definition. See, for example, Kabat, 1991, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927- 948 (Chothia numbering scheme); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86: 9268-9272 (ABS numbering scheme); and Lefranc et al., 2003, Dev. Comp. Immunol. 27: 55-77 (IMGT numbering scheme). Public databases for identifying CDR sequences in antibodies can also be obtained.

Derived from : As used in the text, the term "derived from" refers to the relationship between the first and second molecules. Generally speaking, it refers to the structural similarity between the first molecule and the second molecule and does not imply or include restrictions on the method or source of the first molecule derived from the second molecule.

EC50 : The term "EC50" refers to the half-maximal effect concentration of the antibody or MBM that elicits half of the response between baseline and maximum after a specific exposure time. The EC50 basically represents the concentration of the antibody or MBM where 50% of its maximum effect is observed. In a specific example, the EC50 value, for example, measured by FACS binding analysis, is equal to the concentration of the antibody or MBM that binds to half of the cells expressing the target molecule, and the target molecule can be specifically bound by the antibody or MBM. Therefore, as the EC50 or half-maximum effective concentration value increases, a decreased or weaker binding is observed. In some specific examples, the EC50 value of MBM herein is characterized as having a value of about 10 ^-5 M or lower (for example, lower than 10 ^-5 M, lower than 10 ^-6 M, lower than 10 ^-7 M, lower than 10 ^-8 M, or lower than the EC50 value of ^{10 -9 M).}

Epitope : An epitope or epitope is a part of an antigen (for example, a target molecule) recognized by the antibody or other antigen-binding portion described in the text. Epitopes can be linear or configuration.

Fab : The term "Fab" in the context of MBM herein refers to a pair of polypeptide chains. The first polypeptide system includes the variable heavy chain (VH) region of the antibody N-terminal and the first constant domain (referred to as C1 in the text) The connection to the second polypeptide system includes the connection between the variable light chain (VL) region of the N-terminus of the antibody and the second constant domain (referred to as C2 in the text) that can pair with the first constant domain. In a native antibody, VH is the first constant domain (CH1) of the N-terminal connected heavy chain, and VL is the constant domain (CL) of the N-terminal connected light chain. The Fab herein can be arranged according to the original orientation or include domain substitution or exchange to promote correct VH and VL pairing, especially when the MBM line herein includes different Fabs. For example, it is possible to replace the CH1 and CL domain pairs in the Fab with a pair of CH3-regions to promote correct modified Fab-chain pairing in the heterodimer MBM. It is also possible to reverse CH1 and CL so that CH1 is connected to VL and CL is connected to VH, a configuration generally called Crossmab. Another option, or in addition, the use of substitution or exchange constant domains, by using a universal light chain that can pair with the two variable regions of the heterodimer MBM herein, the correct chain pairing can be achieved.

Fc domain and Fc region : The term "Fc domain" refers to a part of a heavy chain that is paired with a corresponding part of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule, which is formed by the combination of two heavy chain Fc domains. The two Fc domains in the Fc region may be the same or different from each other. The Fc domains in natural antibodies are typically the same, but for the purpose of making the MBM herein, one or two Fc domains can advantageously be modified to allow heterodimerization.

Half antibody : The term "half antibody" refers to at least an ABS or ABS chain (e.g., a Fab chain) and can interact with another molecule including an ABS or ABS chain, such as disulfide bridges or molecular interactions (e.g., Fc hetero Knob-in-hole (knob-in-hole) interaction between dimers) molecules that bind together. Half antibodies can be composed of one polypeptide chain or more than one polypeptide chain (for example, two polyether chains of Fab). In a preferred embodiment, the semi-antibody system includes an Fc domain.

Host cell : The term "host cell" as used herein refers to a cell into which the nucleic acid herein has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably in the text. Please understand that these terms refer to a specific subject cell and the progeny or potential progeny of this cell. Because certain modifications may occur due to mutations or environmental influences in the subsequent generations, these progeny may in fact be different from the parental cells, but they are still included in the scope of the terms as used in the text. A typical host cell is a eukaryotic host cell, such as a mammalian host cell. Exemplary eukaryotic host cells include yeast and mammalian cells, for example, vertebrate cells such as mouse, rat, monkey, or human cell lines, for example, HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

Multispecific binding molecule or MBM: The term "multispecific binding molecule" or "MBM" as used herein refers to a molecule that includes two half antibodies (for example, a combination of multiple polypeptide chains) and is different from at least two Epitopes (and in some cases three or more epitopes) specifically bind to and include ABS1 and ABS2 and ABS3.

Operablely linked: The term "operably linked" as used herein refers to a functional relationship between two or more regions of a polypeptide chain, wherein the two or more regions are connected to produce a functional polypeptide.

Parental antibody : The term "parental antibody" refers to the antibody (the term as defined in the text) from which the MBM herein is derived, for example, there is a parental monospecific or dual-specific MBM lacking the N-terminal scFv region in the MBM herein. Specific antibodies. The MBM herein, for example, in its one or more ABSs, can share binding sequences with the "parental antibody", such as CDR, VH and/or VL sequences, but not necessarily by modifying the parental antibody or its coding sequence Prepared.

Single-chain Fv or scFv : The term "single-chain Fv" or "scFv" as used herein refers to a polypeptide chain that includes the VH and VL domains of an antibody, where these regions exist as a single polypeptide chain.

Specific ( or selective ) binding : The term "specific (or selective) binding" as used herein means that MBM or its antigen binding site ("ABS") forms a complex with the target molecule, which under physiological conditions is Quite stable. Wherein specific binding may be from about 5x10 ^-2 M or less (e.g., less than 5x10 ^-2 M, less than 10 ^-2 M, less than 5x10 ^-3 M, less than 10 ^-3 M, less than 5x10 ^{- 4} M, less than 10 ^-4 M, less than 5x10 ^-5 M, less than 10 ^-5 M, less than 5x10 ^-6 M, less than 10 ^-6 M, less than 5x10 ^-7 M, less than 10 ^-7 M, less than 5x10 ^-8 M, less than 10 ^-8 M, less than 5x10 ^-9 M, less than 10 ^-9 M or less than 10 ^-10 M) KD. Methods for determining the binding affinity of antibodies or antibody fragments, such as MBM or ABS, and target molecules are well known in the art and include, for example, balanced dialysis, surface plasma resonance (such as Biacore analysis), flow cytometry (fluorescent- activated cell sorting, FACS) combined analysis and the like. However, MBM or its ABS antibody that specifically binds to target molecules from one species may have cross-reactivity with target molecules from one or more other species.

Target molecule : The term "target molecule" as used herein refers to any biomolecule (eg, protein, carbohydrate, lipid or combination thereof) that can be specifically bound by the antigen binding site of MBM and expressed on the cell surface.

Tissue : The term "tissue" as used in the text refers to the collection or aggregation of specific types of cells. Tissues can be derived from the mesoderm (e.g., bone, muscle, connective tissue, kidney and related structures), endoderm (e.g., lungs, other respiratory structures, and digestive organs), or ectoderm (e.g., nervous system, sensory organs, skin, and Related structure). Examples of target tissues that can be used as the MBM herein include, but are not limited to, connective tissue (including fibrous connective tissue, skeletal connective tissue and body fluid connective tissue, blood, bone, tendon, ligament, fat and ganglion-like connective tissue, etc.), muscle tissue (Including visceral muscle and smooth muscle, skeletal muscle and heart muscle, etc.), peripheral nerve tissue (cranial nerve, spinal nerve, motor neuron, etc.), epithelial tissue (including skin, trachea, reproductive tract, digestive tract, single-layer squamous epithelium, Stratified squamous epithelium, monolayer cubic epithelium, shifted epithelium, pseudo-stratified columnar epithelium, columnar epithelium, glandular epithelium, ciliated columnar epithelium, etc.), endothelial tissue (blood vessels, lymphatic vessels, etc.), and any cell Or any of its components (for example, extracellular matrix). The target tissue of MBM herein can be (a) solid tissue or liquid tissue (for example, blood or individual blood cell types); (b) normal tissue or diseased tissue (for example cancer cells); and/or (c) The same organ (for example, kidney or liver) or other body structure (for example, tumor) is present or different organs or other body structure are present.

Tissue performance profile : the term "tissue performance profile" as used in the text refers to the expression pattern of the target molecule in the human body, for example, such as the human protein profile (HPA) and/or genotype-tissue performance (GTEx) meter The definition of painting. The tissue performance profile may be a protein performance profile and/or an mRNA performance profile.

Trivalent : The term "trivalent" as used herein refers to an MBM with three antigen binding sites. In some specific examples, two of the antigen binding sites bind to the same epitope of the same target. In other specific examples, two of the antigen binding sites specifically bind to different epitopes of the same target molecule.

Universal light chain : The term "universal light chain" as used herein, in the case of MBM refers to a light chain polypeptide that can pair with the heavy chain region of Fab1 to form Fab1 and can pair with the heavy chain region of Fab2 to form Fab2. Universal light chain is also called "ordinary light chain".

VH : The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, including the heavy chain of scFv or Fab.

VL : The term "VL" refers to the variable region of the immunoglobulin light chain of an antibody, including the light chain of scFv or Fab. 6.2. Multispecific binding molecules (MBM)

The MBM herein includes two half-antibodies, one of which includes at least one antigen-binding site (for example, a Fab) and the other includes at least two antigen-binding sites (for example, having an scFv operably linked to the N-terminus of its VH). Fab).

The MBM herein specifically binds to at least two different epitopes (and in some cases, three or more different epitopes). The at least two different epitopes can be on the same target molecule or on different target molecules. Generally speaking, the MBM herein specifically binds to two or more different target molecules (sometimes referred to as "antigens" in the text). 6.2.1. scFv

Single chain Fv or "scFv" antibody fragments include the VH and VL domains of a single polypeptide chain antibody, which can be expressed as a single chain polypeptide and retain the specificity of the intact antibody derived therefrom. Generally speaking, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form a desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of scFV are the linkers identified in Section 6.2.3.

Unless otherwise indicated, as used herein, scFv may have VL and VH variable regions in any order, for example, in terms of the N-terminus and C-terminus of a polypeptide, scFv may include VL-linker-VH or may include VH- Linker-VL.

This scFv may include VH and VL sequences from any suitable species, such as murine, human or humanized VH and VL sequences.

For the production of scFv-encoding nucleic acids, the VH and VL-encoding DNA fragments are operably linked to another fragment encoding a linker, such as encoding any of the linkers described in section 6.2.3 (typically containing amino acid The repetitive sequence of amino acid and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 4), allows the VH and VL sequences to be expressed as a continuous single-chain protein. The VL and VH domains are represented by Flexible linker connection (see, for example, Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al. , 1990, Nature 348:552-554). 6.2.2. Fab1 and Fab2

The MBM system herein includes at least one Fab region in each half antibody. The Fab region is traditionally produced by the use of enzymes (such as papaya enzyme) by proteolytic cleavage of immunoglobulin molecules. In the MBM herein, this Fab region is recombined and expressed as part of a macromolecule.

This Fab region can include constant and variable region sequences from any suitable species, and therefore can be murine, chimeric, human, or humanized.

The Fab region typically includes the CH1 domain connected to the VH domain, and this region is paired with the CL domain connected to the VL domain. In wild-type immunoglobulins, the VH domain is paired with the VL domain to construct the Fv region, and the CH1 domain is paired with the CL domain to further stabilize the binding module. The disulfide bond between the two constant domains can further stabilize the Fab region.

Regarding the MBM in this article, especially when the light chain is not a common or universal light chain, it is advantageous to use the Fab heterodimerization strategy to allow the Fab regions belonging to the same ABS to bind correctly, and to pair abnormally between the Fab regions belonging to different ABSs. minimize. For example, the Fab heterodimerization strategy shown in Table 1 below can be used: Table 1 Fab heterodimerization strategy Strategy VH CH1 VL CL references CrossMabCH1-CL WT CL domain WT CH1 domain Schaefer et al. , 2011, Cancer Cell 2011; 20: 472-86; PMID: 22014573. Orthogonal Fab VHVRD1CH1CRD2-VLVRD1CλCRD2 39K, 62E H172A, F174G 1R, 38D, (36F) L135Y, S176W Lewis et al. , 2014, Nat Biotechnol 32: 191-8 Orthogonal Fab VHVRD2CH1wt-VLVRD2Cλwt 39Y WT 38R WT Lewis et al. , 2014, Nat Biotechnol 32: 191-8 TCR CαCβ 39K TCR Cα 38D TCR Cβ Wu et al. , 2015, MAbs 7: 364-76 CR3 WT T192E WT N137K, S114A Golay at al. , 2016, J Immunol 196: 3199-211. MUT4 WT L143Q, S188V WT V133T, S176V Golay at al. , 2016, J Immunol 196: 3199-211. DuetMab WT F126C WT S121C Mazor et al. , 2015, MAbs 7: 377-89; Mazor et al. , 2015, MAbs 7: 461-669. Regional exchange WT CH3+knob or hole mutation WT CH3+knob or hole mutation Wozniak-Knopp et al. , 2018, PLoSONE13(4): e0195442

Therefore, in a specific example, the correct binding between the polypeptides of two Fabs can be improved by exchanging the VL and VH domains of the Fab or CH1 and CL with each other, for example, as described in WO 2009/080251.

The correct Fab pairing can also be achieved by introducing one or more amino acid modifications in the CH1 domain or introducing one or more amino acid modifications in the CL domain of the Fab and/or introducing one or more amines in the VH domain Base acid modification and the introduction of one or more amino acid modifications in the VL domain can be improved. The modified amino acid is typically part of the VH:VL and CH1:CL interface, so that the Fab components preferentially pair with each other, rather than with other Fab components.

In a specific example, the one or more amino acid modifications, as indicated by the numbering of Kabat residues, are restricted to conserved framework residues in the variable region (VH, VL) and constant domain (CH1, CL) regions. base. Almagro, 2008, Frontiers In Bioscience 13: 1619-1633 provides definitions of framework residues based on Kaba, Chothia, and IMGT numbering arrangements.

In a specific example, the modifications introduced in the VH and CH1 and/or VL and CL domains are complementary to each other. Complementarity at the interface of the heavy chain and light chain can be achieved on the basis of steric and hydrophobic contact, electrostatic/charge interaction, or a combination of various interactions. The complementarities between protein interfaces are broadly based on the coordination of locks and keys, the knob structure (knob) into the mortar structure (hole), protrusions and cavities, donors and acceptors, etc., described in the literature, all of which mean two The structure of the interaction interface and the nature of the chemical coordination.

In a specific example, the one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In a specific example, the one or more introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary substitutions are described in WO 2014/150973 and WO 2014/082179, the contents of which are incorporated by reference.

In some specific examples, this Fab region includes the 192E substitution in the CH1 domain and the 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, for example, Golay et al. , 2016 , J Immunol 196: 3199-211).

In some specific examples, this Fab region includes the 143Q and 188V substitutions in the CH1 domain and the 113T and 176V substitutions in the CL domain, which are used to exchange the hydrophobic and polar regions of contact between the CH1 and CL domains (see, For example, Golay et al. , 2016, J Immunol 196: 3199-211).

In some specific examples, this Fab region can include modifications in some or all of the VH, CH1, VL, and CL domains to introduce the orthogonal Fab interface to improve the correct assembly of the Fab region (Lewis et al., 2014 Nature Biotechnology 32: 191-198). In a specific example, 39K and 62E modifications are introduced into the VH domain, H172A and F174G modifications are introduced into the CH1 domain, 1R, 38D, (36F) modifications are introduced into the VL domain, and L135Y and S176W modifications are introduced into the CL domain. . In another specific example, the 39Y modification is introduced into the VH domain and the 38R modification is introduced into the VL domain.

The Fab region can also be modified with engineered disulfide bonds to replace the original CH1:CL disulfide bonds, thereby increasing the efficiency of Fab component pairing. For example, engineered disulfide bonds can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, for example, Mazor et al. , 2015, MAbs 7:377-89).

The Fab region can also be modified by replacing the CH1 and CL domains with replacement regions that promote correct assembly. For example, Wu et al. , 2015, MAbs 7:364-76, described replacing the CH1 domain with the constant domain of the T cell receptor, and replacing the CL domain with the b domain of the T cell receptor, and introduced the VL domain The 38D modification in the VH domain and the 39K modification in the VH domain are paired with these regions with additional charge-charge interactions between the VL and VH domains.

Alternatively, or in addition, using a Fab heterodimerization strategy to promote the correct VH-VL pairing, the VL of the ordinary light chain (also known as the universal hydrogen chain) can be used in each Fab VL domain of the MBM herein. In various specific examples, compared with the original homologous VL, the application of the ordinary hydrogen chain as described in the text reduces the number of inappropriate MBM species. In various specific examples, the VL domain of MBM is identified from a monospecific antibody that includes a common hydrogen chain. In various specific examples, the VH domain of MBM includes human heavy chain variable gene fragments rearranged in mouse B cells in vivo, which have been previously engineered to represent a restricted human light chain repertoire or a single human The light chain, which is homologous to the human heavy chain and reacts with the antigen of interest, produces an antibody repertoire containing a plurality of human VH homologous to one or two of the possible human VLs, wherein the antibody repertoire is for the antigen of interest The line is specific. Ordinary light chains are light chains derived from rearranged human VK1-39JK5 sequences or rearranged human VK3-20JK1 sequences, and include somatic mutant (for example, affinity matured) versions. See, for example, U.S. Patent No. 10,412,940. 6.2.3. Linker

In a specific aspect, this document provides MBM in which two or more ABS components (e.g., scFv VH and VL), two or more ABS (e.g., half antibody scFv and Fab), or ABS and non- The ABS components (for example, the Fc region) are connected to each other by a peptide linker. In contrast to ADC linkers used to connect drugs to MBM (such as those described in section 6.4), these linkers are referred to as "ABS linkers" in the text.

The length of the peptide linker can range from 2 amino acids to 60 or more amino acids, and in a specific aspect, the length of a peptide linker ranges from 3 amino acids to 50 amino acids , From 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, from 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids.

In a specific aspect, the peptide linker, for example, the peptide linker that separates the scFv region and the heavy chain, such as the scFv region of ABS1 and the heavy chain variable region of ABS2, has a length of at least 5 amino acids and at least 6 amines. The base acid or at least 7 amino acids and the length as required is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids.

In some of the foregoing specific examples, the length of the linker ranges from 5 amino acids to 50 amino acids, for example, the length ranges from 5 to 50, from 5 to 45, from 5 to 40, and from 5 To 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids. In the foregoing other specific examples, the length of the linker ranges from 6 amino acids to 50 amino acids, for example, the length ranges from 6 to 50, from 6 to 45, from 6 to 40, and from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids. In the foregoing other specific examples, the length of the linker ranges from 7 amino acids to 50 amino acids, for example, the length ranges from 7 to 50, from 7 to 45, from 7 to 40, and from 7 to 40. To 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids.

Charged (for example, charged hydrophilic linker) and/or flexible linker are particularly preferred.

Examples of flexible ABS linkers that can be used in the MBM herein include those described by Chen et al. , 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al. , 2014, Protein Engineering, Design & Selection 27 (10): Examples disclosed in 325-330. Particularly useful flexible linkers are or include repeated glycine and serine, such as _{monomers or multimers of G n} S (SEQ ID NO: 2) or SG _n (SEQ ID NO: 3), Wherein n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a specific example, the linker is or includes a repeating G ₄ S (SEQ ID NO: 4), such as a monomer or multimer of _{(GGGGS) n.}

Polyglycine linkers can be suitable for MBM herein. In some specific examples, the peptide linker, such as the peptide linker that separates the scFv region and the heavy chain, such as the scFv region of ABS1 and the heavy chain variable region of ABS2, includes two consecutive glycine ( 2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly (SEQ ID NO: 5)), five consecutive glycines (5Gly (SEQ ID NO: 6)), six consecutive Glycine (6Gly (SEQ ID NO: 7)), seven consecutive glycines (7Gly (SEQ ID NO: 8)), eight consecutive glycines (8Gly (SEQ ID NO: 9)) or nine consecutive Glycine (9Gly (SEQ ID NO: 10)).

In a specific example, the ABS linker, for example, the peptide linker that separates the scFv region and the heavy chain, such as the scFv region of ABS1 and the heavy chain variable region of ABS2, is composed of G ₄ S (SEQ ID NO: 4) or a multimer thereof and one or more additional glycines, such as 2Gly, 3Gly or 4Gly (SEQ ID NO: 5). Examples of such linkers include G ₄ S GG (SEQ ID NO: 63), 4xG ₄ S GG (SEQ ID NO: 64), and 7xG ₄ S GG (SEQ ID NO: 65). 6.2.4. Hinge area

The MBM herein can also include, for example, the hinge region connecting the ABS module and the Fc region. This hinge region can be a native or modified hinge region. The hinge region is typically found at the N-terminus of the Fc region.

The native hinge region is the hinge region generally found between the Fab and Fc regions in naturally-occurring antibodies. The modified hinge region is any hinge that is different in length and/or composition from the original hinge region. Such hinges may include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions may include complete hinge regions derived from the heavy chain Fc region of antibodies of different types or subtypes. Alternatively, the modified hinge region may include a part of the natural hinge or the repeating unit in which the repeating unit is derived from the natural hinge region. In another alternative, the natural hinge region can be changed by converting one or more cysteine or other residues into neutral residues, such as serine or alanine, or by adding appropriate The inserted residues are converted into cysteine residues to change. By this method, the number of cysteine residues in the hinge region can be increased or decreased. Other modified hinge regions can be completely synthetic or can be designed to have desired properties, such as length, cysteine composition, and elasticity.

Many modified hinge regions have been described in, for example, U.S. Patent No. 5,677,425, WO 99/15549, WO 2005/003170, WO 2005/003169, WO 2005/003170, WO 98/25971 and WO 2005/003171 and these patents It is incorporated into the text by way of reference.

In a specific example, one or two of the half antibodies herein have a complete hinge region, such as a hinge domain, at their N-terminus. In some specific examples, the "hinge domain" refers to a sequence from about Glu216 or about Cys226 to about Pro230 of human IgG1 (Burton, 1985 Molec. Immunol. 22:161-206), or another antibody class or isotype The corresponding sequence.

In various specific examples, the positions 233-236 in the hinge domain can be G, G, G, and gaps; G, G, gaps and gaps; G, gaps, gaps, and gaps; or all gaps, where the position number is Numbered in EU.

In some specific examples, the MBM system herein includes a modified hinge domain, which has a reduced binding affinity for an Fcγ receptor relative to a wild-type hinge domain of the same isotype (for example, human IgG1 or human IgG4).

In a specific example, the Fc region of one or both chains of the MBM herein has a complete hinge domain at its N-terminus.

In a specific example, the two Fc regions and hinge region of MBM herein are derived from lgG4 and the hinge region includes the modified sequence CPPC (SEQ ID NO: 11). Compared with lgG1 containing the sequence CPPC (SEQ ID NO: 11), the core hinge region of human lgG4 contains the sequence CPSC (SEQ ID NO: 12). The presence of serine residues in the lgG4 sequence increases the elasticity of this region, and therefore a certain proportion of molecules form disulfide bonds (intrachain disulfide bonds) in the same protein chain, rather than with another heavy chain in the IgG molecule Bridging to form interchain disulfide bonds (Angel et al. , 1993, Mol Immunol 30(1): 105-108). The serine residue was replaced with proline to obtain the same core sequence as lgG1, which allowed the formation of interchain disulfide bonds in the hinge region of lgG4, thus reducing the heterogeneity of the purified product. This changed isotype line is called lgG4P. 6.2.4.1. Chimeric hinge sequence

This hinge region may be a chimeric hinge region.

For example, a chimeric hinge may include a combination of the "upper hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4 and the "lower hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4 .

In a specific embodiment, the chimeric hinge region includes the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 13) (previously disclosed as SEQ ID NO: 8 of WO2014/121087, which is incorporated herein by reference in its entirety) Middle) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 14) (previously disclosed as SEQ ID NO: 9 of WO2014/121087). These chimeric hinge sequences can be appropriately linked to the IgG4 CH2 domain (for example, by incorporating an IgG4 Fc domain, such as a human or murine Fc domain, it can be further modified in the CH2 and/or CH3 domain to reduce effector Function, for example, as described in section 6.2.5.1). 6.2.4.2. Hinge sequence with reduced effector function

In another specific example, the hinge region can be modified to reduce effector function, for example, as described in WO2016161010A2 (which is incorporated herein by reference in its entirety). In various specific examples, the positions 233-236 of the modified hinge region are G, G, G and gaps; G, G, gaps and gaps; G, gaps, gaps and gaps; or all are gaps, where the position number is With EU number (as shown in Figure 1 of WO2016161010A2). These fragments can be represented by GGG-, GG--, G--- or ----, where "-" represents a gap.

Position 236 is empty in standard human IgG2, but it is occupied in other standard human IgG isotypes. In all four human isotypes, positions 233 to 235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

Hinge modifications in positions 233 to 236 can be combined with position 228 occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but by S in human IgG4 and by R in human IgG3. The S228P mutation in the IgG4 antibody is advantageous in stabilizing the IgG4 antibody and reducing the exchange of the paired heavy chain and light chain between exogenous and endogenous antibodies. Preferably, positions 226 to 229 are occupied by C, P, P, and C, respectively.

The exemplified hinge region has residues 226 to 236, sometimes referred to as the middle (or core) and lower hinge, and is designated by the designation GGG-(233-236), GG--(233-236), G --- (233-236) and no G (233-236) modified hinge sequence occupied. If necessary, the amino acid sequence of the hinge region includes CPPCPAPGGG-GPSVF (SEQ ID NO: 15) (previously disclosed as SEQ ID NO: 1 of WO2016161010A2), and CPPCPAPGG-GPSVF (SEQ ID NO: 16) (previously The disclosed is SEQ ID NO: 2 of WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO: 17) (previously disclosed is SEQ ID NO: 3 of WO2016161010A2), or CPPCPAP---GPSVF (SEQ ID NO: 18) (Previously disclosed is SEQ ID NO: 4 of WO2016161010A2).

The above-mentioned modified hinge region may be incorporated into a heavy chain constant domain, and the heavy chain constant domain typically includes CH2 and CH3 domains, and may have additional hinge segments (e.g., upper hinge) flanking the designated region. Although these additional constant domain fragments may be hybrids of different homotypes, they are typically of the same isotype, preferably the human isotype. The isotype of these other human constant domains is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3 or hybrids thereof, wherein the regions are of different isotypes. Illustrative human IgG1, IgG2 and IgG4 sequences are shown in Figures 2-4 of WO2016161010A2.

In specific embodiments, the modified hinge sequence can be linked to the IgG4 CH2 domain (for example, by incorporating an IgG4 Fc domain, such as a human or murine Fc domain, which can be further modified in the CH2 and/or CH3 domain, Used to reduce effector functions, for example, as described in section 6.2.5.1). 6.2.5. Fc domain

The MBM herein can include an Fc region derived from any suitable species. In a specific example, this Fc region is derived from a human Fc domain.

This Fc domain can be derived from any suitable class of antibodies, including IgA (including lgA1 and lgA2 subtypes), IgD, IgE, IgG (including lgG1, lgG2, lgG3 and lgG4 subtypes), and IgM. In a specific example, the Fc domain is derived from lgG1, lgG2, lgG3, or lgG4. In a specific example, the Fc domain is derived from lgG1. In a specific example, the Fc domain is derived from lgG4.

The two Fc domains in the Fc region may be the same or different from each other. In native antibodies, these Fc domains are typically the same, but for the purpose of manufacturing multispecific binding molecules, such as MBM herein, these Fc domains should advantageously be different to allow heterodimerization, As described in section 6.2.5.2 below.

In native antibodies, the heavy chain Fc domain of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domain of IgE and IgM is composed of three heavy chain constant domains (CH2 , CH3 and CH4). These domains dimerize to produce an Fc region.

In the MBM herein, the Fc region and/or the Fc domain within it may include heavy chain constant domains from one or more different classes, such as one, two or three different classes of antibodies.

In a specific example, the Fc region includes CH2 and CH3 domains derived from lgG1.

In a specific example, the Fc region includes CH2 and CH3 domains derived from lgG2.

In a specific example, the Fc region includes CH2 and CH3 domains derived from lgG3.

In a specific example, the Fc region includes CH2 and CH3 domains derived from lgG4.

In a specific example, the Fc region includes the CH4 region derived from IgM. This IgM CH4 region is typically located at the C-terminus of the CH3 domain.

In a specific example, the Fc region includes CH2 and CH3 domains derived from IgG and a CH4 region derived from IgM.

It should be understood that the heavy chain constant domains used to make the Fc region of the MBM herein may include variants of the naturally occurring constant domains as described above. Compared to the wild-type constant domain, these variants may include one or more amino acid variations. In one example, the Fc region herein includes at least one constant domain that changes in sequence from a wild-type constant domain. It should be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In other examples, the constant domains of these variants are at least 70% identical or similar. In other examples, the constant domains of these variants are at least 80% identical or similar. In other examples, the constant domains of these variants are at least 90% identical or similar. In other examples, the constant domains of these variants are at least 95% identical or similar.

IgM and IgA are naturally produced in humans and are covalent polymers of common H2L2 antibody units. When it is incorporated into the J-chain, IgM is formed as a pentamer, or when it lacks a J-chain, it is a hexamer. IgA is produced as a monomer or dimer. The heavy chains of IgM and IgA have 18 amino acids extending to the C-terminal constant domain, called tailpieces. The tail piece includes cysteine residues, which form disulfide bonds between the heavy chains of the polymer, and it is believed that it plays an important role in the multimerization. The tail piece also contains a glycosylation site. In certain specific examples, the MBM in this article does not include trailers.

The Fc domain of the MBM incorporated herein may include one or more modifications that alter the functional properties of the protein, such as binding to Fc-receptors, such as FcRn or leukocyte receptors, binding to complement, modifying the structure of disulfide bonds, or changing sugars. Basic model. Examples of Fc modifications that alter effector function are described in section 6.2.5.1.

Fc domains can also be modified to include, for example, modifications that improve the manufacturability of asymmetric MBM by allowing heterodimerization, where heterodimerization is such that different Fc domains are paired preferentially over identical Fc domains. Heterodimerization allows the manufacture of MBM, in which different ABSs are connected to each other by Fc regions containing different Fc domains in sequence. Examples of heterodimerization strategies are illustrated in section 6.2.5.2.

It should be understood that any of the above-mentioned modifications can be combined in any suitable manner to achieve the desired functional characteristics, and/or combined with other modifications to change the properties of MBM. 6.2.5.1. Fc domain with altered effector function

In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to Fc receptors and/or effector functions.

In a particular embodiment, the Fc receptor is an Fcγ receptor. In a specific example, the Fc receptor is a human Fc receptor. In a specific example, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRlla, and most specifically human FcγRllla. In a specific example, the effector function is selected from one or more of the following groups: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis ( ADCP), and cytokine secretion. In a particular embodiment, this effector function is ADCC.

In a specific example, the Fc region includes an amino acid substitution selected from the group of positions: E233, L234, L235, N297, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc region includes an amino acid substitution selected from the group of positions: L234, L235, and P329 (numbered according to the Kabat EU index). In some specific examples, this Fc region includes L234A and L235A amino acid substitutions (numbered according to the Kabat EU index). In this specific example, the Fc region is an Igd Fc region, especially a human Igd Fc region. In a specific example, the Fc region includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid is substituted with P329A or P329G, especially P329G (according to the Kabat EU index number). In a specific example, the Fc region includes an amino acid substitution at position P329 and another amino acid substitution selected from the following positions: E233, L234, L235, N297 and P331 (numbered according to the Kabat EU index). In a more specific embodiment, this additional amino acid is substituted with E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, this Fc region includes amino acid substitutions at positions P329, L234, and L235 (numbered according to the Kabat EU index). In a more specific embodiment, this Fc region includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA" or "LALAPG").

Typically, the two Fc domains of the Fc region each have the same one or more amino acid substitutions. Therefore, in a specific example, each Fc domain of the Fc region includes L234A, L235A, and P329G amino acid substitutions (Kabat EU index numbers), that is, in each of the first and second Fc domains of the Fc region, the position The leucine residue at 234 was replaced by alanine residue (L234A), the leucine residue at position 235 was replaced by alanine residue (L235A) and the proline residue at position 329 was replaced by glycine Residue substitution (P329G) (numbered according to Kabat EU index).

In a specific example, the Fc domain is an IgG1 Fc domain, specifically a human IgG1 Fc domain.

Typically, the two Fc domains of the Fc region each have the same one or more amino acid substitutions. Therefore, in a specific example, each Fc domain of the Fc region includes L234A, L235A, and P329G amino acid substitutions (Kabat EU index numbers), that is, in each of the first and second Fc domains of the Fc region, the position The leucine residue at 234 was replaced by alanine residue (L234A), the leucine residue at position 235 was replaced by alanine residue (L235A) and the proline residue at position 329 was replaced by glycine Residue substitution (P329G) (numbered according to Kabat EU index).

In a specific example, the Fc domain is an IgG1 Fc domain, especially a human IgG1 Fc domain. In some specific examples, the IgG1 Fc domain is a variant IgG1 that includes D265A and N297A mutations (EU numbering) to reduce effector functions.

In another specific example, the Fc domain is an IgG4 Fc domain with reduced Fc receptor binding. An exemplary IgG4 Fc domain with reduced Fc receptor binding may include an amino acid sequence selected from Table A below. In some specific examples, this Fc domain only includes the bolded part of the sequence shown below: Table A Fc region sequence SEQ ID NO : SEQ ID NO: 1 of WO2014/121087 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 19 SEQ ID NO: 2 of WO2014/121087 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 20 SEQ ID NO: 30 of WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys twenty one SEQ ID NO:31 of WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys twenty two SEQ ID NO: 37 of WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys twenty three SEQ ID NO: 38 of WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys twenty four

In a specific example, the IgG4 line with reduced effector function includes the bolded part of the amino acid sequence of SEQ ID NO: 31 of WO2014/121087, sometimes referred to as IgG4 or hIgG4 in the text.

For the heterodimer MBM, it is possible to incorporate the aforementioned variant IgG4 Fc sequence, for example, including SEQ ID NO: 30 of WO2014/121087 (or its bold black font part) and SEQ ID NO: 37 of WO2014/121087 (or The Fc region of the bold font part), or the Fc including the combination of SEQ ID NO: 31 of WO2014/121087 (or the bold black font part) and SEQ ID NO: 38 of WO2014/121087 (or the bold black font part) Area. 6.2.5.2. Fc heterodimerization variants

Many multispecific molecular patterns cause dimerization between the two Fc domains. Unlike native immunoglobulins, they are connected to different antigen-binding regions (or parts thereof, such as VH or VH of Fab). -CH1). Inappropriate heterodimerization of the two Fc regions to form the Fc domain may increase the barrier to the production of the desired multispecific molecule and represents a purification challenge. There are various methods available in this technology that can be used to enhance the dimerization of the Fc domain that may be present in the MBM herein, for example, as disclosed in EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573 ; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application No. 2006204493A1; and PCT Publication No. WO2009/089004A1.

This document provides MBM including Fc heterodimers, that is, Fc regions including heterologous, non-identical Fc domains. The heterodimerization strategy is used to increase the dimerization of the Fc region operably connected to different ABSs (or parts thereof, such as VH or VH-CH1 of Fab), and to reduce the two Fc regions operably connected to the same ABS. Polymerization. Typically, each Fc domain in an Fc heterodimer includes the CH3 domain of an antibody. This CH3 domain is derived from any isotype, type or subtype, and is preferably a constant domain of an antibody of the IgG (lgG1, lgG2, lgG3 and lgG4) class as described in the previous section.

The heterodimerization of two different heavy chains in the CH3 domain produces the desired MBM, while the homodimerization of the same heavy chain will reduce the yield of the desired MBM. Therefore, in a preferred embodiment, the two half-antibodies that combine to form the MBM herein will contain a modified CH3 domain, which prefers heterodimerization binding relative to the unmodified heavy chain.

In a specific example, the modification that promotes the formation of Fc heterodimers is a so-called "knob-into-hole" or "knob-in-hole" modification, and the modification is included in one of the Fc domains. The "knob" modification of the Fc domain and the "hole" modification in the other Fc domain. The knob-into-hole technology is described in US Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7- 15 in. Generally speaking, this method involves the introduction of a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide, so that the protrusion can be positioned in the cavity so that Promote the formation of heterodimers and hinder the formation of homodimers. The overhang is introduced by replacing the small amino acid side chain from the interface of the first polypeptide with a larger side chain (such as tyrosine or tryptophan). By substituting a smaller amino acid side chain (such as alanine or threonine) for a larger amino acid side chain, a complementary cavity with the same or similar size as the protrusion is created at the interface of the second polypeptide.

Therefore, in some specific examples, the amino acid residues in the CH3 domain of the first unit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby in the first unit of A protrusion is generated in the CH3 domain, which can be located in the cavity in the CH3 domain of the second subunit, and the amino acid residue in the CH3 domain of the second subunit of the Fc domain is an amine with a smaller side chain volume The base acid residue is substituted, thereby creating a cavity in the CH3 domain of the second unit, in which the protrusion in the CH3 domain of the first unit can be located. Preferably, the amino acid residue with larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W ). Preferably, the amino acid residue with smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V ). The protrusions and cavities can be made by changing the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

In a specific example, the threonine at position 366 in the first Fc domain is substituted with a tryptophan residue (T366W), and the tyrosine residue at position 407 in the Fc domain is substituted with a valerate residue. ( According to the Kabat EU index number). In another specific example, the serine residue at position 354 in the first Fc domain is substituted with a cysteine residue (S354C) or the glutamine at position 356 is substituted with a cysteine residue. (E356C) (in particular, the serine residue at position 354 is substituted with a cysteine residue), and additionally the tyrosine residue at position 349 in the second Fc domain is substituted with cysteine Residue substitution (Y349C) (numbered according to Kabat EU index). In a specific embodiment, the first Fc domain system includes amino acid substitutions S354C and T366W, and the second Fc domain system includes amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU index).

In some specific examples, electrostatic steering (for example, as described in Gunasekaran et al. , 2010, J Biol Chem 285(25): 19637-46) can be used to enhance the binding of the first and second subunits of the Fc domain.

As an alternative, or in addition, using an Fc domain modified to enhance heterodimerization, the Fc domain can be modified to allow for the selection of purification strategies for Fc heterodimers. In this specific example, the semi-antibody system includes a modified Fc domain that eliminates its binding to protein A, thereby enabling purification methods to produce heterodimerized proteins. See, for example, U.S. Patent No. 8,586,713. Therefore, the MBM system includes a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains are different from each other by at least one amino acid, and compared to the corresponding ABM lacking amino acid differences, wherein the At least one amino acid difference reduces the binding of MBM to protein A. In a specific example, the first CH3 domain binds to protein A and the second CH3 domain contains a mutation/modification that reduces or eliminates protein A binding, such as H95R modification (IMGT exon number; EU number is H435R). The second CH3 may further include a Y96F modification (IMGT; EU is Y436F). Such modifications are referred to in the text as "star" mutations. 6.3. Target molecule

The ABS1, ABS2 and ABS3 of the MBM herein are each specifically bound to target molecules, such as cell surface antigens such as proteins, carbohydrates or lipids. In some specific examples, the target molecules that bind to ABS1, ABS2, and ABS3 are protein molecules. Example target molecules include human klotho beta ("KLB"), human fibroblast growth factor receptor 1c isoform ("FGFR1c"), human fibroblast growth factor receptor 3 (" FGFR3"), human CD63, and human amyloid precursor-like protein 2 (APLP2).

Preferably, the scFv, Fab1 and Fab2 lines are selected so that each of ABS1, ABS2 and ABS3 can specifically bind to their respective targets at the same time. In some specific examples, ABS1, ABS2, and ABS3 each specifically bind to different target molecules. In other specific examples, two of the ABS1, ABS2, and ABS3 can bind to different epitopes on the same target molecule.

When two of the ABS1, ABS2 and ABS3 bind to different epitopes on the same target molecule, the binding with the target molecule is preferably non-competitive, that is, ABS will not compete with the target molecule (which may occur , For example when epitopes overlap). Analysis for measuring the binding competition between antibodies and antibody fragments is known in the art and includes, for example, enzyme-binding immunosorbent assay (ELISA), flow cytometric sorting (FACS) analysis, and surface plasma resonance.

For example, the competition of target molecule binding can be analyzed on the Octet HTX biosensor platform (Pall ForteBio Corp.) using real-time, calibration-free biofilm interference technology. In a specific example of this analysis, the entire analysis was performed at 25°C in 10 mM HEPES buffer, 150 mM NaCl, 3 mM EDTA, 1 mg/mL BSA, 0.05% v/v surfactant, Tween -20, pH 7.4 (HBS-EBT buffer) in a measuring plate oscillating at 1000 rpm. In order to analyze whether the two antibodies or their antigen-binding fragments can compete with each other to bind to individual epitopes on their specific target antigens, firstly, the tip of the sensor is immersed in the target antigen containing 5-His label ("Five -His" is disclosed as SEQ ID NO: 25), the 5-His-labeled target antigen ("五-His" is disclosed as SEQ ID NO: 25) is captured onto the tip of the Octet sensor ( Fortebio Inc, #18-5122) anti-5-His antibody ("Five-His" is disclosed as SEQ ID NO: 25). Then, by immersing the first antibody or its antigen-binding fragment (hereinafter referred to as Ab-1) into the wells (for example, 50 µg/mL solution) of the first antibody or its antigen-binding fragment (hereinafter referred to as Ab-1), the Ab-1 is used to capture the Antigen saturation. The sensor tip is then immersed in a well containing a solution (for example, a 50 µg/mL solution) of the second antibody or its antigen-binding fragment (hereinafter referred to as Ab-2). Wash the sensor tip with HBS-EBT buffer between each analysis step. The immediate binding reaction can be monitored throughout the analysis period and the binding reaction can be recorded at the end of each step. It is possible to compare the binding reaction between Ab-2 and the target antigen previously complexed with Ab-1 and determine the competition/non-competition behavior of different antibodies/antigen-binding fragments to the same target antigen.

MBM that binds at least two or more different target molecules can be used to preferentially target specific tissues on which two or more target molecules are expressed, while minimizing binding to other tissue types that are not desired to bind. For example, when one or two of ABS1, ABS2, and ABS3 are combined with the first target molecule with the first tissue performance map, and at least one of ABS1, ABS2, and ABS3 is a second tissue that overlaps but is different from the first performance map When the expression profile is different from the second target molecule, the MBM can target the normal tissues of the first and second expression profiles.

MBM with more than one ABS that binds to the same target molecule (whether in the same epitope or different epitopes) can be used to cluster and activate cell surface receptors.

Performance profiles can be determined based on experience or obtained from public databases, such as the Genotypic Tissue Expression (GTEx) Project and Human Protein Atlas. Target selection can be based on protein expression profiles, mRNA expression profiles, or both. Minimize MBM activity at undesired tissue locations. The tissue performance overlap between two or all three ABS1, ABS2 and ABS3 is preferably less than 10 tissues, for example, 9 tissues, 8 tissues, and 7 One organization, 6 organization, 5 organization, 4 organization, 3 organization, 2 organization or 1 organization. The tissue map analysis showing the example is provided as an example of the target pair binding to MBM, KLB and FGFR1c in this paper, which is shown in Figure 4A (based on the GTEx database) and 4B (based on the HPA database).

In various specific examples: • ABS1 and ABS2 bind to the same or different epitopes on the same target molecule while ABS3 binds to different target molecules; • ABS1 and ABS3 bind to the same or different epitopes on the same target molecule. ABS2 binds to different target molecules; ABS2 and ABS3 bind to the same or different epitopes on the same target molecule, while ABS1 binds to different target molecules; ABS1, ABS2, and ABS3 bind to different target molecules; or ABS1 , ABS2 and ABS3 bind to the same or different epitopes on the same target molecule.

When two or more of ABS1, ABS2, and ABS3 bind to the same epitope on the target molecule, the ABS may have the same or different VH sequence and/or VL sequence.

Without being limited by theory, it is believed that the MBM herein has the advantage of binding to target molecules (or cells that express a target molecule) with greater affinity than parental monospecific antibodies or bispecific antibodies lacking all three ABSs. Therefore, the MBM herein can be greater than the parental monospecific antibody or bispecific antibody lacking all three ABSs in certain specific examples, for example, the affinity of the bispecific antibody lacking ABS1 scFv and one or more target molecules and / Or cell binding of one or more target molecules. _{For example, MBM may have a lower K D} that binds to the target molecule in certain specific examples and/or has a stronger potency than the corresponding parental monospecific antibody or bispecific antibody in a cell-binding-based analysis. The EC50 value (for example, as described in section 7).

The agonist or antagonist activity of a particular antibody or MBM depends on the target selection, epitope coverage, and the type of selection. The identification of agonist and antagonist antibodies can be performed, for example, through function-based screening. When the parental antibody or the parental bispecific antibody has agonist or antagonist activity before scFv fusion, the N-terminal scFv MBM herein has agonist or antagonist activity. Without wishing to be bound by theory, it is believed that when compared to traditional bispecific molecules (for example, a parental bispecific molecule containing two Fabs but lacking the N-terminal scFv region), the MBM herein is, for example, due to the additional The new binding stoichiometry conferred by the N-terminal scFv region has the properties of increased activity (for example, agonist or antagonist activity). 6.3.1. KLB-FGFR1c binder

The MBM herein may contain one or more ABSs that bind to human KLB and one or more ABSs that bind to human FGFR1c (for example, D2 or D3 loop) in some specific examples. Human tissues exhibiting both KLB and FGFR1c include adipose tissue, breast tissue, liver, lung, pancreas, stomach, and testicles (see, Figures 4A to 4B). Therefore, MBMs herein with one or more ABSs that bind to human KLB and one or more ABSs that bind to human FGFR1c can preferentially target these tissue types. FGF21, a member of the FGF family, transmits signals via a receptor complex composed of FGFR1c and KLB and is therefore an endocrine hormone. In an example configuration of this target pair, ABS1 and ABS3 bind to different epitopes of KLB and ABS2 binds to FGFR1c. Without being bound by theory, it is believed that binding MBM with this configuration agonizes the receptor complex and causes the metabolism as shown in FIG. 5.

Exemplary anti-KLB antibody systems are provided in Table 2A and the VH and VL sequences of exemplary anti-KLB antibodies that can be used in the MBM herein are provided in Table 2B. The MBM herein can include, for example, the CDR or VH and/or VL sequences of any anti-KLB antibody provided in Table 2A or Table 2B. Exemplary anti-FGFR1c antibody systems are provided in Table 3A and the VH and VL sequences of the exemplified anti-FGFR1c antibodies that can be used in the MBM herein are provided in Table 3B. The MBM herein may include, for example, the CDR or VH and/or VL sequences of any anti-FGFR1c antibody provided in Table 3A or Table 3B. Table 2A KLB binder name references Sequences in patent references 39F7 (Amgen) US 8,263,074; J. Biol. Chem. (2018) 293, 14678 Heavy chain: Seq ID No. 82; Light chain: Seq ID No. 27 mimAb1 (Amgen) US 2011/0135657; Sci. Transl. Med. (2012) 4, 162ra153 8C5.K4H3.M4L.KNV (Genentech) US 2015/0218276 Heavy chain: Seq ID No. 129; Light chain: Seq ID No. 131 5H23_Humanization (NGM) US 9,738,716 Heavy chain: Seq ID No. 317; Light chain: Seq ID No. 319 Table 2B KLB binder and VL sequences -VH Binder chain sequence SEQ ID NO : 39F7 (Amgen) VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWVAVIWYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRAAAGLHYYYGMDVWGQGTTVTVSS 26 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGSSPLTFGGGTEVEIK 27 5H23 (NGM) VH (vH3) QVQLQQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWIGWIYPGDGSTKYNEKFKGKATITRDTSASTAYMELSSLRSEDTAVYFCARSDYYGSRSFAYWGQGTLVTVSS 28 VL (vL2) DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYVYMHWYQQKPGQPPKLLIYLASYLESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSRDLTFPFGGGTKLEIK 29 Table 3A FGFR1c binder name gauge references Sequences in patent references YW182.5_YGDY (Genentech) D2 ring US 2015/0218276 Heavy chain: Seq ID No. 133; Light chain: Seq ID No. 135 R1Mab1 (Genentech) D2 ring US 9,085,626 Heavy chain: Seq ID No. 2; Light chain: Seq ID No. 6 R1Mab2 (Genentech) D2 ring US 9,085,626 Heavy chain: Seq ID No. 3; Light chain: Seq ID No. 6 R1Mab3 (Genentech) D2 ring US 9,085,626 Heavy chain: Seq ID No. 4; Light chain: Seq ID No. 6 FR1-H7 (ImClone/Eli Lilly) D2 ring US 8,263,074 Heavy chain: Figure 1A; Light chain: Figure 1B FR1-A1 (ImClone/Eli Lilly) D3 ring US 8,263,074 Heavy chain: Figure 2A; Light chain: Figure 2B Table 3B FGFR1c binder -VH and VL sequences Binder chain sequence SEQ ID NO : FR1-H7 (ImClone/Eli Lilly) VH EVQLVQSGAEVKKPGASVKVSCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARDDYMDVWGKGTLVTVSS 30 VL ETTLTQSPDTLSLSPGEGATLSCRASQSVSGSALAWYQQKPGQAPRLLIYDASSRATGVPDRFSGSGSGADFSLTISRLEPEDFAVYSCQQYGSSPLTFGPGTKVDVK 31 FR1-A1 (ImClone/Eli Lilly) VH QVQLVQSGAEVKKPGSSVKVSCKASGQTFTGYYMHWVRQAPGQGLEWMGRIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGDLGGMDVWGQG 32 VL EIVLTQSPLSLPVTPGEPASISCRSSQSLRHSNGYNYLDWYLQKPGQSPQLLIYLASNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQIPPTFGPGTKVDK 33 R1Mab1 (Genentech) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTWISWVPGKGLEWVGEIDPYDGDTYYADSVKGRFTISADTSKNLQMNSLRAEDTAVYYCASSGYGGSDYAMDYWGQ 34 VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKWEIK 35 R1Mab2 (Genentech) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSNNYIHWVPGKGLEWVADIYPNDGDTDYADSVKGRFTISADTSKNLQMNSLRAEDTAVYYCAREHFDAWVHYYVMDYWGQ 36 VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKWEIK 35 R1Mab3 (Genentech) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTSNWISWVPGKGLEWVAEIDPYDGATDYADSVKGRFTISADTSKNLQMNSLRAEDTAVYYCATGTDWMDYWGQ 37 VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKWEIK 35 6.3.2. FGFR3-APLP2 binder

The MBM herein may contain one or more ABSs that bind to human FGFR3 and one or more ABSs that bind to human APLP2 in some specific examples. FGFR3 is a clinically proven carcinogenic driver of bladder cancer. APLP2 has been identified as a cell surface receptor that can undergo rapid internalization and degradation. The MBM herein may have one or more ABSs that bind to human FGFR3 and one or more ABSs that bind to human APLP2 to trigger FGFR3 blockade and increase FGFR3 downregulation via APLP2. In an example configuration of this target pair, ABS2 and ABS3 bind to the same epitope of FGFR3 and ABS1 binds to APLP2. Without being bound by theory, it is believed that binding to MBM with this configuration causes blockage and/or degradation of the FGFR3 receptor complex.

Exemplary anti-FGFR3 antibody sequences are provided in Table 4. The MBM herein may include, for example, the CDR or VH and/or VL sequences of any of the anti-FGFR3 antibodies provided in Table 4. Exemplary anti-APLP2 anti-systems are provided in Table 5. The MBM herein may include, for example, the CDR or VH and/or VL sequences of any of the anti-APLP2 antibodies provided in Table 5. Table 4 FGFR3 binder Binder chain sequence SEQ ID NO : Vofatamab (Rainier) HC EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTGISWVRQAPGKGLEWVGRIYPTSGSTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTYGIYDLYVDYTEYVMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 38 LC DIQMTQSPSSLSASVGDRVTITCRASQDVDTSLAWYKQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSTGHPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSTGHPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALLIYSASFLYSGVQSLGEVSSNRGSLGSLGSLVSSQSGSLGSLVSSV 39 IMC-D11 (Eli Lilly/ImClone) VH EVQLVQSGAEVKKPGASVKVSCKASGYMFTSYGISWVRQAPGQGLEWMGWVSTYNGDTNYAQKFQGRVTVTTDTSTSTAYMELRSLRSEDTAVYYCAR VLGYYDSIDGYYYGMDVWGQGTTVTVSS 40 VL QSVLTQPPSLSVAPGKTATFTCGGNNIGDKSVHWYRQKPGQAPVLVMYLDTERPSGIPERMSGSNFGNTATLTITRVEAGDEADYYCQVWDSGSDHW FGGGTKLTVLG 41 Table 5 APLP2 binders Binder chain sequence SEQ ID NO : H4xH21362P2 (Regeneron) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYVMSWVRQAPGKGPEWVSGISGRTGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASRITAAGRGYYYYYGMDVWGRGTTVTVSS 42 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK 43 H4xH21387P2 (Regeneron) VH EVQLVESGGGLVQPGRSLRLSCVASGFTFADYAMHWVRQAPGKGLEWVSGISWNSGNIDYADSVKGRFTISRDNAKNSLYLQMNSLRTEDTALYYCAKVRIVVAGYYYYYYGMDVWGQGTTVTVSS 44 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK 43 H4xH21371P2 (Regeneron) VH QVQLVQSGVEVKKPGASVKVSCKASGYTFTDYGISWVRQAPGQGLEWMGWISAHNGNTNYAQKLQGRVTMTTDTSTNTAYMELRSLRSDDTAVYYCARRNWKYFDYWGQGTLVTVSS 45 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK 43 6.3.3. FGFR3-CD63 binder

The MBM herein may contain one or more ABSs that bind to human FGFR3 and one or more ABSs that bind to human CD63 in some specific examples. FGFR3 is a clinically proven carcinogenic driver of bladder cancer. CD63 is a four transmembrane protein on the cell surface that can regulate the general performance of the transport of binding partners. The MBM herein may have one or more ABSs that bind to human FGFR3 and one or more ABSs that bind to human CD63 to trigger FGFR3 blockade and promote FGFR3 downregulation or surface retention via CD63. In the example configuration of this target pair, ABS2 and ABS3 bind to the same epitope of FGFR3 and ABS1 binds to CD63. Without being bound by theory, it is believed that binding to MBM with this configuration causes blockage and/or degradation of the FGFR3 receptor complex.

Exemplary anti-FGFR3 antibody sequences are provided in Table 4 above. The MBM herein may include, for example, the CDR or VH and/or VL sequences of any of the anti-FGFR3 antibodies provided in Table 4. The VH and VL sequences of anti-CD63 antibodies that can be used in the examples of MBM herein are provided in Table 6. The MBM herein may include, for example, the CDR or VH and/or VL sequences of any of the anti-CD63 antibodies provided in Table 6. Table 6 CD63 binding promoter sequences and VL -VH Binder chain sequence SEQ ID NO : H5C6 VH EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMSWVRQTPEKRLEWVAYISSSGGSTYYSDTVKGQFTISRDNAKNTLYLQMSRLKSEDTAMYYCARREDYDGRLTYWGQGTLVTISA 46 VL DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMNWYQQKPGQPPKVLIYLASKLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIK 47 NVG-2 VH EVQLVESGGGLVQPGKSLKLTCATSGLTFNTAWMHWVRQSPDKRLEWIGRIKDKSNNYAADYVESVRGRFTISRDDSKSSIYLQMNSLKEEDSATYFCFHNSLAYWGQGTMVTVSS 48 VL NIVMTQSPKSMSISVGDRVTMNCKASQNVDNSIAWYQQKPGQSPKLLIYYATNRYTGVPDRFTGGGFGTDFTLTISSVQPEDAASYYCQRIYDCPNTFGGGTKLELK 49 H1M12451N (Regeneron) VH QVQLQESGPGLMKPSETLSLTCTVSGGSFSSYYWNWIRQSPGKGLEWIGYIRYSGDTNYKPSLKSRFTISIDTSKNLFSLRLKSVTAADTAVYYCARMGLGSDAFDIWGQGTMVTVSS 50 VL EIVLTQSPGTLSLSPGERATLSCRASQSVNNNYLAWYQQKPGQAPRLLIYGVFNRATNIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK 51 H2M12395N (Regeneron) VH QVQLQESGPRLVKPSETLSLTCIVSGGSISNFYWNWIRQSPGKGLEWIGYFFYTGTIDYNPSLKSRVTISLDTSKNQFSLNLRLLTAADAAVYYCARMGLGANAFDIWGHGTMVTVSS 52 VL EIVLTQSPGTLSLSPGERATLSCRASQHVSSNYLAWYQQKPGQAPRLLIYGGSSRATGIPDRFSGSGSGSGTDFTLTISRLEPADFAVFYCQQYGNSPWTFGQGTKVEMK 53 H4H12450N (Regeneron) VH QVQLQESGPKVVKPSETLSLTCTVSGGSISSYYWNWIRQSPGKGLEWIGYTKRGYTDYNPSLRSRVTISEDTSKNQFSLRISSVTAADTAVYYCAQMGWGSHAFDMWGQGTMVAVSS 54 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK 55 H2M12450N (Regeneron) VH QVQLQESGPKVVKPSETLSLTCTVSGGSISSYYWNWIRQSPGKGLEWIGYTKRGYTDYNPSLRSRVTISEDTSKNQFSLRISSVTAADTAVYYCAQMGWGSHAFDMWGQGTMVAVSS 54 VL EIVLTQSPGTLSLSPGERATLSCRASQSVNSRYLAWYQQKPGQAPRLLIYGASSRATGIPDRCSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK 56 6.4. Antibody drug conjugate

The MBM herein can, for example, be joined to a drug group via a linker, especially when the MBM is intended to be used as a cancer therapeutic agent. For convenience, these conjugates are referred to herein as antibody-drug conjugates (or "ADCs").

In a specific aspect, the drug group exerts cytotoxic or cytostatic activity. In a specific example, the drug group is selected from the group consisting of maytansinoid, kinesin-like protein KIF11 inhibitor, V-ATPase (vacuolar-type H+-ATPase) inhibitor, cell promoting Apoptosis agent, Bcl2 (B-cell lymphoma 2) inhibitor, MCL1 (myelogenous leukemia 1) inhibitor, HSP90 (heat shock protein 90) inhibitor, IAP (inhibitor of apoptosis) inhibitor, mTOR ( Mechanism targets of rapamycin) inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, and MetAP (methionine amino peptidase) , CRM1 (Chromosome Maintenance Protein 1) Inhibitor, DPPIV (Dipeptidyl Peptidase IV) Inhibitor, Proteasome Inhibitor, Inhibitor of Phosphoryl Transfer Reaction in Mitochondria, Protein Synthesis Inhibitor, Kinase Inhibitor, CDK2 (Cyclin-dependent kinase 2) inhibitors, CDK9 (Cyclin-dependent kinase 9) inhibitors, kinesin inhibitors, HDAC (histone deacetylase) inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalation Agents, DNA minor groove binders, RNA polymerase inhibitors, topoisomerase inhibitors or DHFR (dihydrofolate reductase) inhibitors.

.In some specific examples, the cytotoxic agent is a maytansinoid with the following structure:

.

.In some specific examples, the cytotoxic agent is a maytansinoid with the following structure:

.

其中

為連接MBM之鍵結。In some specific examples, this ADC includes the MBM and

in

It is the bond connecting MBM.

In some specific examples, the antibody-drug conjugate system includes the MBM herein, and

其中

為連接MBM之鍵結。

in

It is the bond connecting MBM.

，或

，或 其混合物， 其中

為連接本文MBM之鍵結。In some specific examples, this ADC includes the MBM and

,or

, Or a mixture thereof, where

To connect the MBM of this article.

In some specific examples, this bond is connected to the MBM via the sulfur component of the cysteine residue.

In some specific examples, this bond is connected to MBM via the nitrogen component of the lysine residue.

In the ADC herein, the cytotoxic agent and/or cytostatic agent is connected to the MBM through the ADC linker. The ADC linker connecting the cytotoxic agent and/or cytostatic agent and the MBM of the ADC can be short, long, hydrophobic, hydrophilic, elastic or rigid, or can be composed of fragments each independently having one or more of the above properties , So that the linker can include fragments with different properties. This linker can be multivalent, allowing it to covalently link more than one reagent to a single position on the MBM, or monovalent, enabling it to covalently link a single reagent to a single position on the MBM.

In a specific aspect, the linker is selected from a cleavable linker, a non-cleavable linker, a hydrophilic linker, a precharged linker, or a dicarboxylic acid-based linker.

Those skilled in the art should understand that the ADC linker connects the cytotoxic agent and/or by forming a covalent bond of the cytotoxic agent and/or cytostatic agent at one position and the covalent bond of the MBM at another position. Cell inhibitors and MBM. This covalent bond is formed by the functional group on the ADC linker and the reaction between the agent and the functional group on MBM.

The ADC linker is preferably, but not necessarily, chemically stable to adapt to the outside of the cell, and can be designed to be lysed, destroyed, and/or otherwise particularly degraded within the cell. Alternatively, ADC linkers that are not designed for intracellular lysis or degradation can be used. The choice of stable and unstable ADC linker can be determined according to the toxicity of the cytotoxic agent and/or cytostatic agent. For reagents that are toxic to normal cells, a stable linker is preferred. Reagents that are selective or targeted and less toxic to normal cells can be used, and the ADC linker is less important for the chemical stability of the extracellular environment. Various ADC linkers that can be used to connect drugs to MBM are known in the art. Any of these ADC linkers, as well as other ADC linkers, can be used to connect the cytotoxic agent and/or cytostatic agent to the MBM of the ADC herein.

Exemplary multivalent ADC linkers that can be used to link many cytotoxic agents and/or cytostatic agents to a single MBM molecule are described in, for example, WO 2009/073445; WO 2010/068795; WO 2010/138719; WO 2011/120053; WO 2011/171020; WO 2013/096901; WO 2014/008375; WO 2014/093379; WO 2014/093394; WO 2014/093640, the content of which is incorporated herein by reference in its entirety. For example, the Fleximer linker technology developed by Mersana et al. has the potential to obtain high-DAR ADCs with good physical and chemical properties. The Mersana technology is based on the incorporation of drug molecules into the dissolved polyacetal backbone via a series of ester bonds. This method gives high-load ADCs (DAR up to 20) while maintaining good physical and chemical properties.

Exemplary monovalent ADC linkers that can be used are described in, for example, Nolting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045: 71-100; Ducry et al., 2010, Bioconjugate Chem. 21: 5-13; Zhao et al., 2011, J. Med. Chem. 54: 3606-3623; U.S. Patent No. 7,223,837; U.S. Patent No. 8,568,728; U.S. Patent No. 8,535,678; and WO2004010957, each of which is incorporated herein by reference middle.

By way of example and without limitation, certain cleavable and non-cleavable ADC linker systems that can be included in the ADC herein are described below.

In a specific embodiment, the ADC linker selected is cleavable in vivo. The cleavable ADC linker may include chemically or enzymatically unstable or degradable linkages. Cleavable ADC linkers generally rely on intracellular processing to release drugs, such as reduction in the cytoplasm, exposure to acidic conditions of the lysosome, or cleavage by specific proteases or other enzymes in the cell. The cleavable ADC linker generally incorporates one or more chemically or enzymatically cleavable chemical bonds, while the rest of the ADC linker is non-cleavable. In a specific embodiment, the ADC linker system includes a chemically labile group, such as a hydrazone and/or a disulfide group. The linker system that includes chemically labile groups takes advantage of the differential properties between plasma and certain cytoplasmic compartments. The intracellular conditions for promoting drug release of the ADC linker containing hydrazone are the acidic environment of endosomes and lysosomes, while the ADC linker containing disulfide groups is in the presence of high thiol concentration, such as glutathione. Reduction in cytoplasmic fluid. In a specific embodiment, the plasma stability of the ADC linker including a chemically unstable group can be increased by using a substituent close to the chemically unstable group and introducing a steric barrier.

The cleavable ADC linker may include a non-cleavable portion or fragment, and/or the cleavable fragment or portion may be included in another non-cleavable ADC linker to impart cleavability. Merely by way of example, polyethylene glycol (PEG) and related polymers can include cleavable groups in the backbone of the polymer. For example, polyethylene glycol or polymer ADC linkers may include one or more cleavable groups, such as disulfide bonds, hydrazones, or dipeptides.

Other cleavable linkages that may be included in the ADC linker include ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on the bioactive agent, wherein these ester groups Generally, it is hydrolyzed under physiological conditions to release the bioactive agent. Hydrolytically degradable linkages include, but are not limited to, carbonic acid linkages; imine linkages produced by the reaction of amines and aldehydes; phosphate linkages formed by the reaction of alcohols and phosphoric acid groups; condensation of the reaction products of aldehydes and alcohols Aldehyde linkage; orthoester linkage of the reaction product of formic acid and alcohol; and oligonucleotide linkage formed by phosphoamidite groups, including (but not limited to) at the end of the polymer and the oligonucleotide 5'hydroxyl group.

In a specific embodiment, the ADC linker system includes an enzyme-cleavable peptide group, such as a tripeptide or dipeptide. In a specific embodiment, the dipeptide system is selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu -Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Val-Lys; Ala-Lys; Phe-Cit ; Leu-Cit; lle-Cit; Phe-Arg; and Trp-Cit. In a specific embodiment, the dipeptide system is selected from: Cit-Val; and Ala-Val.

In any of the various specific examples of ADCs discussed above or herein, the ADC may have a drug:antibody ratio (or in this case, a drug:MBM ratio) ranging from 1 to 20, and more typically ranging from 2 to 10. 6.5. Nucleic acids and host cells

In another aspect, this document provides a nucleic acid encoding the MBM herein. In some specific examples, this MBM is encoded by a single nucleic acid. In other specific examples, the MBM is encoded by a plurality of nucleic acids (for example, two, three, or four) nucleic acids.

A single nucleic acid can encode an MBM that includes a single polypeptide chain, an MBM that includes two or more polypeptide chains, or a part of an MBM that includes two or more polypeptide chains (for example, a single nucleic acid can encode three, four, or more Two polypeptides of the MBM of the polypeptide chain, or three polypeptides of the MBM including four or more polypeptide chains). With regard to individual control of performance, an open reading frame encoding two or more polypeptide chains can be under the control of individual transcriptional regulatory elements (e.g., promoters and/or enhancers). The open reading frame encoding two or more polypeptide chains can also be controlled by the same transcriptional regulatory elements and separated by internal ribosome entry site (IRES) sequences for translation into individual polypeptides.

In some embodiments, the MBM including two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding MBM may be equal to or lower than the number of polypeptide chains in MBM (for example, when more than one polypeptide chain is encoded by a single nucleic acid).

The nucleic acid herein can be DNA or RNA (e.g., mRNA).

In another aspect, this document provides host cells and vectors containing the nucleic acid herein. This nucleic acid may be in a single vector or in a separate vector in the same host cell, as explained in more detail below. 6.5.1. Carrier

This document provides a vector comprising a nucleotide sequence encoding the MBM or MBM component described herein, such as one or two polypeptide chains of a half antibody. This carrier system includes, but is not limited to, virus, plastid, mucilage, lambda phage or yeast artificial chromosome (YAC).

There are many vector systems available. For example, one of the vector systems utilizes DNA elements derived from animal viruses, such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous Sarcoma virus (Rous Sarcoma virus) virus), MMTV or MOMLV) or SV40 virus. Another type of carrier system utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern equine encephalitis virus, and yellow fever virus.

In addition, cells that have stably integrated DNA into their chromosomes can be selected by introducing one or more markers that allow the selection of transfected host cells. This label can provide, for example, the transfer of protons to the auxotrophic host, the resistance of microbicides (for example, antibiotics), or the resistance of heavy metals, such as copper or the like. The selectable marker gene can be directly connected to the desired DNA sequence, or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcription promoters, enhancers, and termination signals.

Once the expression vector or DNA sequence containing the structure is ready for expression, the expression vector can be transfected or introduced into an appropriate host cell. This can be accomplished using various techniques, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. The methods and conditions for culturing the transfected cells produced and for recovering the expressed polypeptides are known to those skilled in the art, and according to the specific expression vector and mammalian cells used, based on this specification, Change or optimize. 6.5.2. Cells

Also provided herein are host cells that include the nucleic acids herein.

In a specific example, the host cell line is genetically engineered to include one or more of the nucleic acids described herein.

In a specific example, the host cell line is genetically engineered through the use of expression cassettes. The term "expression cassette" refers to a nucleotide sequence capable of expressing a gene in a host compatible with this sequence. These expression cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors that are necessary or helpful for performance can also be used, such as, for example, inducible promoters.

Also provided herein are host cells that include the vectors described herein.

The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to Sf9 cells. 6.6. Pharmaceutical composition

The MBM and/or ADC herein may be in the form of a composition including MBM and/or ADC and one or more carriers, excipients and/or diluents. The composition can be formulated for specific purposes, such as veterinary medicine or human medicine. The form of the composition (eg, dry powder, liquid formulation, etc.) and the excipients, diluents and/or carriers used will depend on the desired MBM and/or ADC use, and therapeutic use, and the mode of administration. .

For therapeutic use, the composition can be supplied as a part of a pharmaceutical composition that is sterile, including a pharmaceutically acceptable carrier. The composition can be in any suitable form (depending on the desired method of administering it to the patient). The pharmaceutical composition can be administered to the patient through various routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, or topical. The most suitable route of administration in any particular case will depend on the particular antibody and/or ADC, the subject and the nature and severity of the disease, and the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

The pharmaceutical composition can conveniently be presented in a unit dosage form containing a predetermined amount of the MBM and/or ADC of the present disclosure per dose. The amount of MBM and/or ADC included in the unit dose will depend on the disease to be treated and other factors well known in the art. These unit dosage forms may be lyophilized powders containing an amount of MBM and/or ADC suitable for single administration, or may be in liquid form. The dry powder unit dosage form can be packaged as a kit with a syringe, an appropriate amount of diluent, and/or other components that can be used for administration. The unit dose in liquid form can conveniently be supplied in the form of a syringe pre-filled with an amount of MBM and/or ADCU suitable for single administration.

The pharmaceutical composition can also be supplied in bulk form containing the amount of ADC suitable for multiple administrations.

The pharmaceutical composition can be prepared by combining MBM and/or ADC with the desired purity with pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to as "carriers" in the text), as needed, typically used in this technology. ”), that is, buffers, stabilizers, preservatives, isotonic agents, non-ionic detergents, antioxidants, and various other additives are mixed to prepare them for storage as freeze-dried formulations or aqueous solutions. See, Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). These additives should be non-toxic to the recipient in the dosage and concentration used.

Buffers help maintain the pH within the range of approximate physiological conditions. It can be present in a wide variety of concentrations, but will typically be present in a concentration range from about 2 mM to about 50 mM. Buffers suitable for use herein include organic and inorganic acids and their salts, such as citrate buffers (e.g., sodium dihydrogen citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-lemon Sodium dihydrogen acid mixture, etc.), succinate buffer (for example, succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffer (for example, Tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffer (for example, fumaric acid-fumarate monosodium mixture, fumaric acid-fumarate disodium mixture, fumarate monosodium-fumarate disodium mixture, etc.) Etc.), gluconate buffer (for example, gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture, etc.), oxalate buffer (for example, oxalate-sodium oxalate mixture , Oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffer (for example, lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffer (for example, Acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

Preservatives can be added to retard the growth of microorganisms, and can be added in an amount ranging from about 0.2% to 1% (w/v). Preservatives suitable for use herein include phenol, benzyl alcohol, m-cresol, methyl paraben, propyl paraben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride halide ( benzalconium halide) (such as chloride, bromide and iodide), hexamethonium chloride and alkyl p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate, catechol, m- Hydroquinone, cyclohexanol and 3-pentanol. Isotonic agents, sometimes called "stabilizers", can be added to ensure the isotonicity of the liquid composition herein, and include polysaccharide alcohols, such as trivalent or higher sugar alcohols, such as glycerol, erythritol, Arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a wide range of excipients, whose functions range from fillers to dissolving therapeutic agents or additives that help prevent denaturation or stick to the walls. Typical stabilizers can be polysaccharide alcohols (listed above); amino acids, such as arginine, lysine, glycine, glutamic acid, aspartic acid, histidine, alanine, Ornithine, L-leucine, 2-phenylalanine, glutamine, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol , Ribitol, inositol, galactitol, glycerol and the like, including cyclic alcohols, such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, and lipoic acid , Sodium thioglycolate, thioglycerol, α-mercaptoglycerol, and sodium thiosulfate; low molecular weight polypeptides (such as polypeptides with 10 or fewer residues); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immune Globulin; Hydrophilic polymers such as polyvinylpyrrolidone, monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccharides such as cotton seed Sugar; and polysaccharides, such as dextran. Depending on the weight of the ADC, the stabilizer may be present in an amount ranging from 0.5 to 10 wt%.

Non-ionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve glycoproteins and protect glycoproteins from aggregation caused by agitation. It also allows the formulation to be exposed to shear surface stress without causing Protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and pluronic polyols. The nonionic surfactant may be present in the range of about 0.05 mg/mL to about 1.0 mg/mL, for example, about 0.07 mg/mL to about 0.2 mg/mL.

Additional various excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and co-solvents. 6.7. Treatment indications

The MBM, ADC, and pharmaceutical compositions herein can be used to treat cancer, such as cancers related to target molecules that are combined with ABS1, ABS2, and ABS3.

Therefore, in one aspect, this document provides a method of treating cancer, which method comprises administering to a subject suffering from cancer an effective amount of the MBM, conjugate or pharmaceutical composition herein. In a specific example, the MBM herein, which binds to FGFR3, and optionally to CD63 or APLP2, can be used to treat bladder cancer, glioblastoma, and squamous cell lung cancer.

The MBM, ADC and pharmaceutical compositions herein can also be used for non-cancer indications, such as the treatment of non-alcoholic steatohepatitis ("NASH"), the treatment of metabolic diseases, lower circulating HDL cholesterol, increase circulating LDL cholesterol, and lower Blood triglycerides, treatment of obesity and treatment of diabetes. For these non-cancer indications, in some specific examples, MBM is targeted at KLB and/or FGFR1c.

Therefore, in one aspect, this document provides a method of lowering HDL cholesterol in the circulation, the method comprising administering an effective amount of the MBM herein, a conjugate, or a pharmaceutical composition to a subject suffering from an elevated HDL amount.

In another aspect, this document provides a method of increasing circulating LDL cholesterol, the method comprising administering an effective amount of the MBM herein, a conjugate, or a pharmaceutical composition to a subject suffering from a low LDL amount.

In another aspect, this document provides a method for lowering blood triglycerides, the method comprising administering an effective amount of the MBM, conjugate or pharmaceutical composition herein to a subject suffering from an elevated amount of triglyceride.

In another aspect, this document provides a method of lowering blood glucose, the method comprising administering an effective amount of the MBM, conjugate or pharmaceutical composition herein to a subject suffering from an elevated blood glucose level.

In another aspect, this document provides a method of treating obesity, the method comprising administering to a subject suffering from obesity an effective amount of the MBM, conjugate or pharmaceutical composition herein.

In another aspect, this document provides a method of treating diabetes, the method comprising administering an effective amount of the MBM, conjugate or pharmaceutical composition herein to a subject suffering from diabetes. 7. Examples 7.1 Example 1:.. FGFR1c x KLB x KLB 2 + 1 N-scFv MBMs 7.1.1 Materials and Methods The generation 7.1.1.1.MBM

Fusion of various KLB scFvs that bind to KLB epitope 1 (ep1) or epitope 2 (ep2) with the N-terminus of the FGFR1c VH domain from the existing IgG-like bispecific molecule REGN4366, where REGN4366 is based on both FGFR1c and KLB For the subject (Figure 1 and Table 7).

Encoding (i) various KLB scFvs, in the direction of VL (with 100C mutation, Kabat numbering), linker (4xG4S (SEQ ID NO: 57)) and VH (with 44C mutation, Kabat numbering), and then used for ligation scFvs and FGFR1c bind to Fab's different length linkers, (ii) FGFR1c binds to Fab, and (iii) gG1 Fc region with clubbing mutations (S354C, T366W, EU numbering), forming hole-shaped mutations (Y349C, The DNA fragments of T366S, L368A, Y407V, EU numbering) and star mutations (H435R, Y436F, EU numbering) were synthesized by Integrated DNA Technologies, Inc. (San Diego, California) or GenScript (Piscataway, NJ).

According to the standard molecular selection method provided by New England BioLabs Inc., NEBuilder HiFi DNA combo kit (New England BioLabs Inc.) or restriction digestion followed by conjugation was used to manufacture mammalian expression vectors for individual heavy chains. For the performance of FGFR1c/KLB/KLB 2+1 N-scFv MBMs (F1K-scFv1-9), the heavy chain ("Hc1-Knob" and "Hc2-Hole*") and the general light chain DNA are in accordance with the manufacturer's method Co-transfected into Expi293 cells (ThermoFisher Scientific). 50 ml of cell culture medium was collected and purified through HiTrap Protein A FF column (GE Healthcare). For functional confirmation, the selected MBM was enlarged to 200 ml and a series of purification procedures including size exclusion chromatography as the final step were performed. Production and purification of REGN4366 (with KLB-binding Fab and FGFR1c-binding Fab) as an IgG-like bispecific control molecule. 7.1.1.2. HEK293.SREluc.hFGFR1cHS/ hKLB reporter- based analysis

Use HEK293.SREluc.hFGFR1cHS/hKLB cells stably expressing human FGFR1c and KLB and the luciferase reporter gene under the control of a serum response element (SRE)-containing promoter to detect the agonist activity of MBM. Recombinant human FGF21 with a 6xHis tag (SEQ ID NO: 58) was used as a positive control, and the maximum reporter activity from FGF21 was defined as 100% activity. The cells were treated with each MBM or 6xHis-FGF21 for 6 hours, and then subjected to luciferase analysis. In contrast to the maximum activity elicited by FGF21, the percentage of activity elicited by individual MBMs was normalized. A dose response analysis was performed to determine the EC50. 7.1.1.3. Biacore analysis of the combination of hFGFR1c and hKLB

The affinity and mechanism of F1K-scFv6 binding were determined by Biacore analysis. In order to compare the epitope affinity of F1K-scFv6 to KLB and the monovalent affinity of the parental KLB mAb, the antibody capture mode was used. In short, FGFR1c parental mAb 19842, KLB parental mAb 22532P2 and 22393P2, FGFR1c/KLB parental bispecific antibodies REGN4366 and F1K-scFv6 were immobilized on a CM5 chip with an anti-human Fc antibody (REGN2567). Different concentrations of hFGFR1c_V5_6xHis (800–12.5 nM, 4-fold dilution) or hKLB.HA.6xHis (100–1.56 nM, 4-fold dilution) were injected at 50 µL/min for 2 minutes and analyzed at 25°C. Use 1:1 binding model Use Scrubber 2.0c to measure binding kinetic parameters by fitting real-time data. 7.1.1.4. Cell-based integration of flow cytometry

HEK293/Cas9-hFGFR1/hKLB (KLB+), HEK293/hFGFR1c (hFGFR1c+) and HEK293/hFGFR1c/hKLB (hFGFR1c+/hKLB+) cells were suspended in complete Dulbecco's modified Eagle's medium (complete ^{) at 1×10 6 cells/mL} Dulbecco's Modified Eagle Medium) (DMEM) (10% fetal bovine serum (FBS), 1x penicillin-streptomycin-glutamic acid) and ^{stained with 1×10 5} cells. Add MBM and stain the cells for 30 min at 2 to 8°C. The cells were washed twice with FACS washing buffer (phosphate buffered saline (PBS) containing 1% FBS and 1 mM EDTA) and centrifuged at 1800 RPM for 4 min at 4°C. Add isophycocyanin-conjugated goat anti-human IgG (Jackson Immuno Research, 109-136-098, 1:400) and incubate the cells at 2 to 8°C for 30 min. The cells were washed as before and suspended in 100 μL of 2% paraformaldehyde. Incubate the cells for 30 min at 2 to 8°C and wash twice. BD LSRFortessaTM FACS instrument was used to analyze the stained cells. 7.1.2 Results 7.1.2.1. FGFR1c / KLB / KLB 2 + 1 N-scFv MBM generation of

Expression and purification of nine FGFR1c/KLB/KLB 2+1 N-scFv MBM with the characteristics shown in Table 7. Table 7 Molecular ID Hc1- knob (Hc1-Knob) Hc2- holes * (Hc2-Hole *) Lc 1. KLB scFv Linker 2. FGFR1c 3. KLB FlK-scFv1 22414P2(KLB ep1) 3xG ₄ S (SEQ ID NO: 1) 19842 22393P2 (KLB ep1) General Lc FlK-scFv2 22401P2(KLB ep1) FlK-scFv3 22532P2(KLB ep2) FlK-scFv4 22414P2(KLB ep1) 6xG ₄ S (SEQ ID NO: 59) FlK-scFv5 22401P2(KLB ep1) FlK-scFv6 22532P2(KLB ep2) FlK-scFv7 22414P2(KLB ep1) 9xG ₄ S (SEQ ID NO: 60) FlK-scFv8 22401P2(KLB ep1) FlK-scFv9 22532P2(KLB ep2) Molecular ID Hc1 Hc2* Lc REGN4366 Na Na 19842 22393P2 (KLB ep1) General Lc ep: epitope; *: star mutation 7.1.2.2. Epitope- dependent activity of FGFR1c/KLB/KLB 2+1 N-scFv trispecific molecule

The cell-based reporter analysis results of the agonist activity of the purified FGFR1c/KLB/KLB 2+1 N-scFv MBM using HEK293.SREluc.hFGFR1cHS/hKLB are shown in Table 8. Based on activity, F1K_scFv3, F1K_scFv6 and F1K_scFv9 were found to be the best activators. They all share the same KLB targeting scFv binding to the KLB ep2 region, and in this analysis are significantly more active than the MBM with scFv targeting the same KLB ep1 region as the native KLB Fab. Notably, it is observed that F1K_scFv6 with a cFv-Fab 1 linker with a length of 30 amino acids has the highest activity and best potency of 54.1% (EC50 = 9.80E-10 M). Table 8 The activation of the reporter activity of 2+1 N-scFvs in the analysis based on HEK293.SREluc.hFGFR1cHS/hKLB Molecular ID 1.KLB scFv 2.FGFR1c 3. KLB (FGF21) Activity % EC50 (M) REGN1945 NA NA NA ND ND 6xHis-hFGF21 NA NA NA 100 3.40E-09 F1K_scFv1 22414P2 (KLB ep1) 19842 22393P2 5 3.90E-09 F1K_scFv2 22401P2 (KLB ep1) 19842 22393P2 6.2 3.70E-08 F1K_scFv3 22532P2 (KLB ep2) 19842 22393P2 28.9 7.90E-09 F1K_scFv4 22414P2 (KLB ep1) 19842 22393P2 4.7 3.50E-09 F1K_scFv5 22401P2 (KLB ep1) 19842 22393P2 3 4.70E-08 F1K_scFv6 22532P2 (KLB ep2) 19842 22393P2 54.1 9.80E-10 F1K_scFv7 22414P2 (KLB ep1) 19842 22393P2 5.5 3.60E-09 F1K_scFv8 22401P2 (KLB ep1) 19842 22393P2 4.2 4.20E-08 F1K_scFv9 22532P2 (KLB ep2) 19842 22393P2 20.7 2.40E-09 The activity% represents the relative maximum luciferase activity of each molecule normalized to the value of 6xHis-FGF21. ND: Not detected 7.1.2.3. 2+1 N-scFv trispecific F1K-scFv6 can simultaneously bind to two different epitopes on the same KLB

The Biacore analysis results of F1K-scFv6 are shown in Tables 9A to 9B. It was found that F1K-scFv6 has a KD of 1.47E-11 M for hKLB, which represents a 55-fold and 1,333-fold increase in affinity compared to the parental KLB mAbs 22532P2 and 22393P2, respectively. When F1K-scFv6 was compared with REGN4366 (compared to the 22393P2 arm of hKLB), an increased affinity for hKLB was also observed. This observation strongly supports the result that F1K-scFv6, which has different epitope targeting arms for KLB, can occupy two binding sites on the same hKLB molecule at the same time. In terms of hFGFR1c binding, compared to its parental FGFR1c mAb 19842 or parental bispecific antibody REGN4366 (with the 19842 arm of hFGFR1c), F1K-scFv6 has a slightly lower affinity (4-fold) for hFGFR1c. Table 9A hFGFR1c 6xHis binding kinetics Captured mAb Subject mAb capture volume (RU) 100nM antigen binding (RU) k _a (1/Ms) k _d (1/s) K _D (M) 19842 IgG FGFR1c 677 ± 2.3 107 4.93E+04 2.60E-02 5.27E-07 22532P2 IgG KLB 546 ± 1.6 -8 NB NB NB 22393P2 IgG KLB 553 ± 1 -6 NB NB NB REGN4366 FGFR1c/KLB 605 ± 1.6 50 4.59E+04 2.47E-02 5.39E-07 F1K_scFv6 FGFR1c/KLB/KLB 649 ± 3.1 17 2.10E+04 4.43E-02 2.11E-06 TABLE 9B hKLB.HA.6xHis binding kinetics Captured mAb Target mAb capture volume (RU) 100nM antigen binding (RU) k _a (1/Ms) k _d (1/s) K _D (M) 19842 IgG FGFR1c 670 ± 2.2 -8 NB NB NB 22532P2 IgG KLB 540 ± 1.6 480 1.46E+05 1.20E-04 8.23E-10 22393P2 IgG KLB 547 ± 2 688 5.98E+05 1.17E-02 1.96E-08 REGN4366 FGFR1c/KLB 597 ± 2.4 411 4.76E+05 1.22E-02 2.55E-08 F1K_scFv6 FGFR1c/KLB/KLB 639 ± 3.1 473 1.08E+06 1.59E-05 1.47E-11 7.1.2.4. 2+1 N-scFv trispecific F1K-scFv6 binds more closely to cells overexpressing hKLB and hFGFR1c/hKLB

In order to further confirm the correlation between F1K-scFv6 and the increased affinity of hKLB under the cell-based setting, FACS binding analysis was used to combine the trispecific F1K-scFv6 (FGFR1c/KLB/KLB) with the bispecific antibody REGN4366 (FGFR1c/ KLB 22393 arm) and REGN679 (FGFR1c/KLB 22532 arm), parental antibody 22393 IgG (KLB), 22532 IgG (KLB) and 19842 IgG (FGFR1c) for comparison. In HEK293/Cas9-hFGFR1/hKLB (hFGFR1 knockout and hKLB overexpression) and HEK293/hFGFR1c/hKLB (hFGFR1c and hKLB overexpression), F1K-scFv6 consistently showed stronger binding than all other controls The EC50 (Figures 6A and 6C). In HEK293/hFGFR1c cells (with bivalency to hFGFR1c), 19842 IgG was observed to have the strongest binding, followed by REGN4366 and F1K-scFv6 (Figure 6B). FACS combined data is completely consistent with Biacore analysis. Without being bound by theory, it is believed that the data of this example shows that the bi-epitope binding of hKLB through the 2+1 N-scFv trispecific design enhances the antibody-mediated KLB/FGFR1c receptor complex interaction and potential cell surface Cluster. 7.1.2.5. 2+1 N-scFv MBM F1K-scFv6 has superior agonist activity than bispecific REGN436

In order to evaluate whether the increased affinity of F1K-scFv6 can be translated into a higher efficiency than bispecific REGN4366, HEK293.SREluc.hFGFR1cHS/hKLB was used for in vitro reporter-based cell analysis. After affinity purification, both F1K-scFv6 and REGN4366 were purified by size exclusion chromatography as the final step to remove any aggregates that might disturb the interpretation of the data. Compared to the parental bispecific antibody REGN4366 with only 19% activation, when normalized under the background of soluble FGF21, F1K-scFv6 not only increased the efficiency of luciferase gene expression (expressed in EC50) by 4 to 5 times, but also significantly Increase the% of activation to 77% (Figure 8).

A similar trivalent molecule that uses the same 22532P2 scFv, but is fused to the C-terminus of Fc, is also manufactured and evaluated, called 2+1 C-scFv. This corresponding trispecific molecule has worse performance and purification yield, and worse functional activity than F1K-scFv6 (results not shown). 7.2. Example 2 : Evaluation of linker length to trispecific activity 7.2.1. Materials & methods

By sequentially transfecting HEK293 cells with SRE-luciferase reporter, full-length human FGFR1c and full-length human KLB plastids, a stable cell line HEK293.SREluc.hFGFR1c.hKLB was generated.

The linker length variant of the three-specific molecule called scFv6 was generated and detected by luciferase reporter analysis. The same was done for the specific molecule of the control pair. The linker variants are scFv6 LK7: G4S GG (SEQ ID NO: 63), scFv6 LK15: 3xG4S (SEQ ID NO: 1), scFv6 LK22: 4xG4S GG (SEQ ID NO: 64), scFv6: 6xG4S (SEQ ID NO : 59), scFv6 LK37: 7xG4S GG (SEQ ID NO: 65) and scFv6 LK45: 9xG4S (SEQ ID NO: 60).

The cells were implanted in a 384-well plate and cultured overnight in a complete medium containing 10% fetal bovine serum (FBS). The culture was changed to Opti-MEM reduced serum medium (ThermoFisher, USA) supplemented with 0.1% FBS. After about 24 hrs, the cells were treated with serially diluted ligands for 6 hrs, and then the ONE-Glo™ luciferase analysis system (Promega, USA) was used to perform luciferase analysis according to the manufacturer's instructions. 7.2.2. Results

In one study, the agonist activity of bispecific binding molecules (BBM) called REGN4304 and REGN4366 was compared with the agonist activity of hFGF21. In this analysis, BBM showed approximately 30% of hFGF21 agonist activity (Figure 7).

In another analysis, the agonist activity of FGF21 and the linker length variant of BBM called REGN4304 (the peptide linker between the 2 and 3 regions indicated in Figure 9) were compared. The results are shown in Figure 9 and Table 10 below: Table 10 1 2 3 Linker EC50 %Act REGN1438 6His-FGF21 5.7E-10 100.0 F1K_scFv6 LK7 22393 ADI-19842 22532 L20H7 1.7E-09 104.5 F1K_scFv6 LK15 22393 ADI-19842 22532 L20H15 1.6E-09 97.5 F1K_scFv6 LK22 22393 ADI-19842 22532 L20H22 1.3E-09 87.9 F1K_scFv6 LK30 22393 ADI-19842 22532 L20H30 2.0E-09 84.2 F1K_scFv6 LK37 22393 ADI-19842 22532 L20H37 1.1E-09 80.4 F1K_scFv6 LK45 22393 ADI-19842 22532 L20H45 1.5E-09 78.7 REGN4304 1.8E-09 42.0

All linkers exhibited at least about 2x activity of the control dual-characteristic binding molecule, and the variant with the shortest linker length (7 or 15 amino acids) showed the best agonist activity. 7.3. Example 3 : Activation of FGFR1c signal transmission in HEK293 cells 7.3.1. Materials & Methods

The HEK293.SREluc.hFGFR1c.hKLB stable cell line was generated as described in Example 3. For western blot analysis, HEK293.SREluc.hFGFR1c.hKLB cells were implanted into 6-well plates and cultured in complete medium containing 10% fetal bovine serum (FBS) overnight. The culture was changed to Opti-MEM reduced serum medium (ThermoFisher, USA) supplemented with 0.1% FBS. After approximately 24 hr, add the diluted ligand to the cells to a final concentration of 1 nM or 10 nM. After 15 minutes, the cells were washed with cold PBS and then dissociated in RIPA dissociation buffer (150 mMTris/HCl, pH 7.4, 50 mM NaCl, 1% NP-40 and 0.1% Tween 20). The total cell dissociated substance was re-dissolved by SDS-PAGE and transferred to PVDF membrane. For Western blot analysis, the following primary antibodies were used: total ERK (Cell Signaling, 9102), phosphorylated-ERK (Cell Signaling, 9101), PLC-γ (Cell Signaling, 5690), phosphorylated-PLCγ (Cell Signaling, 2821). 7.3.2. Results

Use HEK293.SREluc.hFGFR1c.hKLB cells stably expressing human FGFR1c and human KLB, with irrelevant antibody REGN1945 as a negative control and FGF21 (REGN1438) as a positive control to test the agonist activity of bispecific and trispecific binding molecules . After treatment, ERK and PLC-γ phosphorylation (the phosphorylation is triggered by activated FGFR1c) was measured by luciferase activity.

Both F1K_scFv6LK7 and F1K_scFv6L1 strongly induced ERK and PLC-γ phosphorylation at concentrations of 1 nM and 10 nM. Obviously, the amount of phospho-ERK and phospho-PLC-γ in cells treated with F1K_scFv6 or F1K_scFv6LK7 was significantly higher than that of the parent bispecific antibody (REGN4366), FGFR1/KLB agonist bispecific antibody ( REGN4304) or recombinant human FGF21 (REGN1438) treated cells (Figure 10A).

To assess the time course of FGFR1c activation, HEK293.SREluc.hFGFR1c.hKLB cells were treated with ligands for different periods of time and collected for Western blot analysis. The result is shown in Figure 10B. As early as 15 min after treatment with REGN1438, REGN4304 or F1K_scFv6, the ERK activation measured by the amount of phosphorylated-ERK was observed and lasted for up to 6 hours. Compared with REGN1438 or REGN4304, F1K_scFv6 showed higher phosphorylation-ERK in the entire treatment time course. At the 15 min time point, F1K_scFv6 strongly induced phosphorylation-PLCγ, and then gradually decreased over time. 7.4. Example 4 : Asymmetric flow field flow separation combined with multi-angle light scattering (A4F-MALLS) in vitro size analysis of the complex formed between KLB , FGFR1c and binding molecules 7.4.1 Overview

In principle, the three specific binding molecules herein can form different types of complexes with FGFR1c and KLB. In order to determine the type of complex formed, asymmetric flow field flow separation combined with multi-angle light scattering (A4F-MALLS) was used to form between 2+1 N-scFv and 2+1 N-Fab trispecific binding molecules In vitro size analysis of complexes. A4F-MALLS was also used to analyze the complex formed by the control bispecific binding molecule (REGN4304) and the monospecific binding molecule (REGN4661). 7.4.2 Materials & methods 7.4.2.1. A4F-MALLS mobile phase buffer

Prepare mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1); then add the solution to a volume of 5.0 L with HPLC grade water. The final measured buffer pH was 7.0. Filter the mobile phase buffer (0.2 μm) before use. 7.4.2.2. A4F-MALLS

The A4F-MALLS system is composed of Eclipse™ 3+ A4F separation system combined with ultraviolet (UV) diode array detector, Wyatt Technology Dawn HELEOS® II laser light scatterometer (LS) and an Optilab® T-rEX differential Refractive index (RI) detector is composed of Agilent 1200 Series HPLC system. The detectors are connected consecutively in the following order: UV-LS-RI. The LS and RI detectors are calibrated according to the instructions provided by Wyatt Technology.

A defined amount of anti-KLB and anti-FGFR1c multispecific binding molecules were mixed with REGN6424 (recombinant KLB) and REGN6152 (recombinant FGFR1c) and diluted with 1X DPBS, pH 7.4 to produce an isomolar ratio: 0.2 μM multispecific Binding molecule: 0.2 μM REGN REGN6424 or 0.2 μM multispecific binding molecule: 0.2 μM REGN REGN6424: 0.2 μM REGN REGN6152. All samples were incubated at ambient temperature for 2 hours without being filtered and kept at 4°C, and then injected into W350 isolation foil pad (350 μm pad thickness, 2.2 cm pad width) and 10 kDa MWCO regenerated cellulose membrane Eclipse™ short channel. Before injection of each sample, the channel is pre-equilibrated with mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1). Bovine serum albumin (BSA; 2 mg/mL; 10 μg sample load) was injected separately and included as a system suitability control.

The separation method is composed of four steps: injection, focusing, dissolution and channel "flushing" steps. The entire separation method uses A4F-MALLS mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1). Each sample (7 μg) was injected at a flow rate of 0.2 mL/min for 1 min and then focused at a focusing flow rate of 1.0 mL/min for 3 min. The sample was eluted with a fixed cross flow of 3.0 mL/min at a channel flow rate of 1.0 mL/min for 15 minutes, followed by a linear gradient cross flow of 3.0 mL/min to 0 mL/min for 5 minutes. Finally, keep the cross flow at 0 mL/min for another 5 min to flush the channel. Use the same parameter settings to fractionate the BSA. 7.4.2.3. MALL data analysis

其中c為溶質濃度，R(θ,c)為以溶質作為散射角和濃度之函數的過量瑞立比值(Raleigh ratio)，Mw為莫耳質量，P(θ)係描述散射光的角度相依性(就迴轉半徑＜ 50 nm的粒子為~1)，A2為滲透壓擴增中的第二維里係數(因為在稀溶液上進行測量，因此可忽略)及方程式 2 ：

其中n₀ 係代表溶劑折射率，N_A 為亞佛加厥常數，λ0為真空中入射光的波長，及dn/dc係代表溶質的比折射率增加量。The data was analyzed using ASTRA V software (version 5.3.4.14, Wyatt Technology). Fit the data to an equation involving excess scattered light and solute concentration and weight-average molar mass Mw (Kendrick et al., 2001, Anal Biochem. 299(2): 136-46; Wyatt, 1993, Anal. Chim. Acta 272(1): 1-40): Equation 1 :

Where c is the solute concentration, R(θ,c) is the excess Raleigh ratio of the solute as a function of the scattering angle and concentration, Mw is the molar mass, and P(θ) describes the angular dependence of the scattered light (For particles with a radius of gyration <50 nm is ~1), A2 is the second virial coefficient in osmotic pressure amplification (because it is measured on a dilute solution, it can be ignored) and Equation 2 :

Representative solvent-based wherein a refractive index n _0, N _A is a constant Ya Fojia Jue, λ0 is the vacuum wavelength of incident light, and dn / dc lines than the refractive index representative of the amount of solute is increased.

The molar mass of the BSA monomer is used to evaluate the light scattering during data collection and the correction constant of the differential refractive index detector (system suitability check). The relative standard deviation (%RSD) of the average molar mass of BSA measured from UV and RI detectors is ≤ 5.0%.

The normalization coefficient of the light scattering detector, the delay volume between the detectors, and the spectral bandwidth expansion items are calculated from the BSA chromatogram collected under the A4F-MALLS condition. These values are applicable to all other samples to calibrate the data files collected for these items.

The protein conjugate analysis provided by Astra software was used to experimentally determine the dn/dc value and the extinction coefficient at 215 nm. Analyze all protein-protein complex samples using the corrected extinction coefficient and dn/dc value. 7.4.3. Results

Use A4F-MALLS to evaluate several recombinant KLB (REGN6424), recombinant FGFR1c (REGN6152) and several monospecific (REGN4661), bispecific (REGN4304) and trispecific (2+1 N-scFv) binding molecules The relative size distribution of the complex formed. The results are shown in Fig. 11A (for REGN4661), Fig. 11B (for REGN4304) and Fig. 11C (2+1 N-scFv mode). The theoretical molar mass and predicted stoichiometry of possible antibody: antigen complexes are provided in the insets of Figures 11A-11C. As expected, when mixed in an equal molar ratio, the monospecific KLB binding molecule (REGN4661) and KLB form a typical 1:1 (peak 1, ~280 kDa) and 1:2 (peak 2, ~356 kDa) Complex (Figure 11A). Similarly, when the control bispecific binding molecule (anti-KLBxFGFR1c; REGN4304) was mixed with an equal molar amount of KLB, a discrete homogeneous peak with a calculated molar mass of ~280 kDa (peak 1) was observed (Figure 11B ). Based on the calculated molar mass of individual components, peak 1 may represent a 1:1 bispecific: KLB complex. In addition, adding FGFR1c to this mixture produces a wide peak (peak 2) with a calculated molar mass range of ~305-444 kDa, which is generally a 1:1:1 bispecific: KLB: FGFR1c ternary complex The objects match (Figure 11B). The upward trending molar mass at the end of peak 2 indicates that the larger complexes are weakly bound through the KLB-FGFR1c interaction, and may also exist in the solution, but are easily dissociated due to layered separation.

Compared with the control monospecific and bispecific binding molecules, the trispecific binding molecule binds KLB and FGFR1c with a unique, higher-level stoichiometry. When mixed with equal molar amounts of KLB, F1K-scFv6 IgG1 forms a widely discrete homogeneous peak with a molar mass of ~579 kDa (peak 1), which may represent 2 molecules of F1K-scFv6 IgG1 and 2 molecules of KLB Bound complex (2:2 complex; Figure 11C). After adding various amounts of FGFR1c to the mixture, a slightly wider post-dissolution peak (peak 2) with a calculated molar mass of ~607-644 kDa was observed. Peak 2 may represent a mixture of ternary complexes containing 2 molecules of F1K-scFv6 IgG1, 2 molecules of KLB and 1-2 molecules of FGFR1c (2:2:1 and 2:2:2 complexes; Figure 11C).

Figure 12A graphically illustrates the stoichiometry of FGF21 in complex with FGFR1c and KLB. Figure 12B graphically illustrates the selective stoichiometry of various FGF21-antibody binding. The data shown in the article reveals that the three specific binding molecules in this article can bind to KLB and FGFR1c to form a ternary complex with unique stoichiometry compared to the control monospecific and bispecific binding molecules. Compared with bispecific binding molecules, it may cause increased agonist activity. 7.5. Example 5 : FGFR3/APLP2 and FGFR3/CD63 2+1 N-scFv MBM 7.5.1. Materials and methods 7.5.1.1 MBM generation

For the construction of 2+1 N-scFv MBM with scFv fused to the bivalent FGFR3 mAb, 16 APLP2-targeting and three CD63-targeting scFv were individually combined with the heavy chain N- of the FGFR3 parent antibody 30108. End fusion, and the antibody system has effector silent substitution, holes (Y349C, T366S, L368A,, Y407V, EU numbering) and star-shaped (H435R, Y436F, EU numbering) mutations of the modified human IgG4 Fc backbone (Table 11 ). The second heavy chain contains the same 30108 Fab and a modified IgG4 Fc with the same effector silent substitution and knob (S354C, T366W, EU numbering) mutation (Table 11). The general form of the 2+1 N-scFv MBM line is illustrated in Figure 1, and the FGFR3/APLP2 and FGFR3/CD63 2+1 N-scFv MBM lines contain silent substitutions, club and socket mutations, and star mutations. The general form of the 2+1 N-scFv MBM line with a hole mutation in the scFv-containing chain and a clubbing mutation in the lack of the scFv region is illustrated in Figure 3C. The general form of 2+1 N-scFv MBM with a star-shaped mutation in the chain containing scFv is illustrated in Figure 3A.

Encoding (i) various APLP2 or CD63 scFv, with VH (with VH-44C, Kabat numbering), linker (4xG4S (SEQ ID NO: 57)), VL (with VL-100C mutation, Kabat numbering), connected The direction of the daughter (G4S)3 (SEQ ID NO: 1) connects scFv and Fab, (ii) the Fab region of 30108, and (iii) the DNA fragment of the modified human IgG4 Fc (Table 10) was produced by Integrated DNA Technologies, Inc. . (San Diego, California) Synthesized. The mammalian expression vectors of individual heavy chains are combined by NEBuilder HiFi DNA Combination Kit (New England BioLabs Inc.). For expressing FGFR3/APLP2 or CD63 2+1 N-scFv molecules, the heavy chain 1-Hole*, heavy chain 2-Knob and ULC 3-20 DNA were co-transfected into Expi293 cells (ThermoFisher Scientific) according to the manufacturer’s method . 50 ml of cell culture medium was collected and purified through HiTrap Protein A FF column (GE Healthcare). For functional confirmation, the selected MBM was enlarged to 200 ml and a series of purification procedures including size exclusion chromatography as the final step were performed. The 30108 IgG targeted by FGFR3 previously manufactured and purified was used as a control group. 7.5.1.2. Biacore analysis of the combination with the target

Biacore kinetic analysis was performed to evaluate the binding affinity of the two MBMs, 3ASB-5 and 3CSB-2, to individual targets. 7.5.1.3. Hyperplasia analysis

UMUC14 cells with S249C mutation and RT4 cells with FGFR3-TACC3 fusion mutation were used to detect the effect of 2+1 N-scFvs on the proliferation of bladder cancer cells. 7.5.2. Results 7.5.2.1 . Generation of FGFR3/APLP2 and FGFR3/CD63 2+1 N-scFv MBM

The design summary of representative FGFR3/APLP2 and FGFR3/CD63 2+1 N-scFv MBM expressed and purified is shown in Table 11. Table 11 Molecular ID Hc1-Hole* Hc2-Knob Lc 1. APLP scFv Linker 2. FGFR3 3. FGFR3 3ASB-5 21375P2 3xG ₄ S (SEQ ID NO: 1) 30108P2 30108P2 ULC 3-20 Molecular ID Hc1-Hole* Hc2-Knob Lc 1. CD63 scFv Linker 2. FGFR3 3. FGFR3 3CSB-2 H4H12450N 3xG ₄ S (SEQ ID NO: 1) 30108P2 30108P2 ULC 3-20 Molecular ID Hc1 Hc2 Lc H4H30108P2 IgG NA NA 30108P2 30108P2 ULC 3-20 7.5.2.2. FGFR3/APLP2 or CD63 2+1 N-scFv can bind to FGFR3 , APLP2 and CD63

The Biacore kinetic analysis results of 3ASB-5 and 3CSB-2 on individual targets are shown in Tables 12A to 12C. It was found that the two 2+1 N-scFv lines were similar to the parental 30108 IgG control group, with KD=8 nM, and combined with the common target FGFR3. For 3ASB-5, the binding of APLP2 was found to be in the subnemolar concentration range, KD = 8.04E-10 M. For 3CSB-2, it was found that the KD of CD63 is 5.88E-10 M, which is 4 times weaker than the monovalent binding affinity of the parental H4H12450N IgG. In summary, both 3ASB-5 and 3CSB-2 have the expected ability to bind to their targets FGFR3, APLP2 and CD63. Table 12A hAPLP2.mmh binding kinetics Captured mAb Subject mAb capture volume (RU) 100nM antigen binding (RU) k _a (1/Ms) k _d (1/s) K _D (M) 3ASB-5 FGFR3/APLP2 202.6 ± 5.8 26.8 5.27E+04 4.24E-05 8.04E-10 H4H30108P2 IgG FGFR3 326.1 ± 2.2 -0.7 NB NB NB REGN1945 FelD1 289.2 ± 2.9 -0.5 NB NB NB Table 12B hCD63.mmh binding kinetics Captured mAb Subject mAb capture volume (RU) 100nM antigen binding (RU) k _a (1/Ms) k _d (1/s) K _D (M) 3CSB-2 FGFR3/CD63 274.3 ± 3.1 19.5 3.59E+05 2.11E-04 5.88E-10 H4H12450N IgG CD63 754.9 ± 2.9 77.6 5.04E+05 7.15E-05 1.42E-10 H4H30108P2 IgG FGFR3 346.5 ± 6.3 -4.3 NB NB NB REGN1945 FelD1 330.6 ± 6.2 0.5 NB NB NB Table 12C hFGFR3b.mmh binding kinetics Captured mAb Subject mAb capture volume (RU) 100nM antigen binding (RU) k _a (1/Ms) k _d (1/s) K _D (M) 3ASB-5 FGFR3/APLP2 276 ± 2.2 90.0 7.39E+04 6.12E-04 8.28E-09 3CSB-2 FGFR3/CD63 274.3 ± 3.1 72.4 6.66E+04 5.71E-04 8.57E-09 H4H30108P2 IgG FGFR3 346.5 ± 6.3 117.8 8.36E+04 6.46E-04 7.73E-09 REGN1945 FelD1 330.6 ± 6.2 1.8 NB NB NB 7.5.2.3. FGFR3/APLP2 2+1 N-scFv 3ASB-5c04FGFR3/CD63 2+1 N-scFv 3CSB-2 can provide effective proliferative resistance in UMUC14 (S249C) and RT4 (FGFR3-TACC3) cells through different mechanisms Cut off

UMUC14 cells with S249C mutation and RT4 cells with FGFR3-TACC3 fusion mutation were used to detect the effect of 2+1 N-scFv on the proliferation of bladder cancer cells. The parent antibody H4H30108P2 exhibited effective growth inhibition in cells, but the growth inhibition in UMUC14 cells was not ideal. 3CSB-2 and 3ASB-5 showed significant boosting activity in UMUC14 cells, increasing growth inhibition by ~25% at the highest tested dose (100nM) (Figure 13A). In addition, the activities of 2+1 N-scFvs 3CSB-2 and 3ASB-5 maintained high amounts in RT4 cells and were comparable to the parent antibody H4H30108P2 (Figure 13B). Different from the parental FGFR3 antibody, 3CSB-2 and 3ASB-5 were found to block the proliferation of bladder cancer cells driven by different mutations (Figures 13A and 13B.

To further study the mechanism behind FGFR3/APLP2 or FGFR3/CD63 MBM, the receptor degradation effect triggered by antibodies was detected on UMUC14 cells. Interestingly, different mechanisms were observed in FGFR3/APLP2 and FGFR3/CD63 MBM. Compared with untreated cells or cells treated with isotype control antibody (REGN1945) or parental antibody H4H30108P2, FGFR3/APLP2 MBM treatment showed increased receptor degradation caused by antibody (Figure 14). On the contrary, treatment with FGFR3/CD63 MBM does not affect the amount of FGFR3 receptor, which means that the growth inhibitory activity of the bispecific 3CSB-2 MBM is increased by different mechanisms (Figure 14). 8. Specific concrete examples

This article uses the following specific specific examples as examples. 1. A multispecific binding molecule (MBM) comprising: (a) a first polypeptide chain, in the direction from N- to C-terminus, comprising (i) containing the first antigen binding site ("ABS1") The scFv is operably linked to (ii) the first heavy chain region of the first Fab ("Fab1"), and the Fab is operably linked to (iii) the Fc domain; (b) the second polypeptide chain is The N-to C-terminal direction includes (i) a second heavy chain region comprising a second Fab ("Fab2"), and the second Fab is operably linked to (ii) the Fc domain; (c) the third A polypeptide chain including the first light chain and the first heavy chain region paired to form Fab1, where Fab1 includes the second antigen binding site ("ABS2"); and (d) the fourth polypeptide chain, which includes the first The second light chain and the second heavy chain are paired to form Fab2, where Fab2 includes the third antigen binding site ("ABS3"). 2. The MBM of specific example 1, wherein each antigen binding site ("ABS") binds to a different epitope. 3. The MBM of specific example 1 or specific example 2, in which two ABS1, ABS2 and ABS3 specifically bind to different epitopes of the same target molecule. 4. The MBM of any one of specific examples 1 to 3, wherein the scFv, Fab1 and Fab2 can simultaneously specifically bind to their respective targets. 5. As the MBM in any of specific examples 1 to 4, at least one of ABS1, ABS2, and ABS3 specifically binds to the target molecule with the first tissue performance profile, and at least one ABS1, ABS2, and ABS3 is associated with the second tissue The target molecule of the performance profile specifically binds, and the second tissue performance profile is overlapped with the first tissue performance profile, but is not the same. 6. The MBM of specific example 5, wherein the first tissue performance map and the second tissue performance map overlap by 10 or less tissues. 7. The MBM of specific example 6, wherein the first tissue performance map and the second tissue performance map overlap by 5 or less tissues. 8. Such as the MBM of specific example 7, wherein the first tissue performance map and the second tissue performance map overlap by 3 or less tissues. 9. The MBM of any one of specific examples 6 to 8, wherein the first and second tissue performance map lineages are defined by the human protein map (HPA) and/or genotypic tissue expression (GTEx) project. 10. The MBM of any one of specific examples 6 to 9, wherein the first and second tissue expression profiles are protein expression profiles. 11. The MBM of any one of specific examples 6 to 9, wherein the first and second tissue expression profiles are mRNA expression profiles. 12. The MBM of any one of specific examples 1 to 11, wherein the affinity of the MBM to the target molecule of Fab1 is lower than the affinity of the second MBM lacking the MBM of scFv to the target molecule of Fab1, but the second MBM The other parts are the same as the MBM in amino acid sequence. 13. The MBM of any one of specific examples 1 to 12, wherein the scFv is connected to the first heavy chain region via a linker. 14. The MBM of specific example 13, wherein the linker has a length of at least 5 amino acids, at least 6 amino acids or at least 7 amino acids, and optionally up to 30 amino acids and up to 40 amines. Amino acids, up to 50 amino acids or up to 60 amino acids, and in certain specific examples, the linker: (a) The length is from 5 amino acids to 50 amino acids; (b) The length is from 5 amino acids to 45 amino acids; (c) The length is from 5 amino acids to 40 amino acids; (d) The length is from 5 amino acids to 35 amino acids; (e) ) The length is from 5 amino acids to 30 amino acids; (f) The length is from 5 amino acids to 25 amino acids; (g) The length is from 5 amino acids to 20 amino acids; ( h) The length is 6 amino acids to 50 amino acids; (i) The length is 6 amino acids to 45 amino acids; (j) The length is 6 amino acids to 40 amino acids; (k) Length from 6 amino acids to 35 amino acids; (l) Length from 6 amino acids to 30 amino acids; (m) Length from 6 amino acids to 25 amino acids ; (N) Length is 6 amino acids to 20 amino acids; (o) Length is 7 amino acids to 40 amino acids; (p) Length is 7 amino acids to 35 amino acids Acid; (q) length from 7 amino acids to 30 amino acids; (r) length from 7 amino acids to 25 amino acids; (s) length from 7 amino acids to 20 amines Base acid; or (t) The length is 10 amino acids to 60 amino acids. 15. The MBM of specific example 14 or 14(m), wherein the linker has a length of 20 amino acids to 50 amino acids, and if necessary, the linker has a length of 25 to 35 amino acids. 16. The MBM of any one of specific examples 13 to 15, wherein the linker is or includes a _{multimer of G n} S (SEQ ID NO: 61) or SG _n (SEQ ID NO: 62), wherein n is 1 An integer from to 7, where the linker system includes a _{multimer of G 4} S (SEQ ID NO: 4) if necessary. 17. The MBM of any one of specific examples 13 to 16, wherein the linker is or includes two consecutive glycines (2Gly), three consecutive glycines (3Gly), and four consecutive glycines (4Gly ( SEQ ID NO: 5)), five consecutive glycines (5Gly (SEQ ID NO: 6)), six consecutive glycines (6Gly (SEQ ID NO: 7)), seven consecutive glycines (7Gly (SEQ ID NO: 8)), eight consecutive glycines (8Gly (SEQ ID NO: 9)) or nine consecutive glycines (9Gly (SEQ ID NO: 10)). 18. The MBM of any one of specific examples 13 to 17, which includes (a) G _n S (SEQ ID NO: 61) or SG _n (SEQ ID NO: 62) as many specifics (for example, G ₄ S ( SEQ ID NO: 4) multimer, wherein n is an integer from 1 to 7, for example, it is a _{multimer including G 4} S (SEQ ID NO: 4); and (b) one or more additional glycosides Amino acids, such as two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly (SEQ ID NO: 5)), five consecutive glycines (5Gly ( SEQ ID NO: 6)), six consecutive glycines (6Gly (SEQ ID NO: 7)), seven consecutive glycines (7Gly (SEQ ID NO: 8)), eight consecutive glycines (8Gly (SEQ ID NO: 9)) or nine consecutive glycines (9Gly (SEQ ID NO: 10)). 19. As in the MBM of any one of specific examples 1 to 18, at least one of ABS1, ABS2 and ABS3 is related to Membrane-bound antigen specifically binds. 20. As in the MBM of any one of specific examples 1 to 19, wherein ABS1 and ABS3 specifically bind to membrane-bound antigens on the same cell. 21. As in any one of specific examples 1 to 20 MBM, which is a trispecific binding molecule ("TBM"). 22. Such as MBM in any one of specific examples 1 to 21, wherein the first light chain and the second light chain are universal light chains. 23. As specific The MBM of any of Examples 1 to 22, wherein the light chain constant region and the first heavy chain constant region (CH1) of the first Fab or the second Fab are arranged in Crossmab. 24. As in any of the examples 1 to 23 MBM, which includes the Fc heterodimer. 25. The MBM of specific example 24, wherein the Fc domain in the Fc heterodimer, compared to the wild-type Fc domain, includes a mutation in the knob and socket structure. 26. As specific The MBM of example 24 or specific example 25, wherein at least one Fc domain in the Fc heterodimer includes a star mutation compared to the wild-type Fc domain. 27. The MBM of any one of specific examples 1 to 26 , Which is a trivalent MBM. 28. Such as the MBM of any one of specific examples 1 to 27, wherein ABS3 specifically binds to human clostin β ("KLB"). 29. T such as the MBM of specific example 28, wherein ABS3 includes the CDR sequences of anti-KLB antibodies, such as the CDR sequences of any of the KLB binders described in Tables 2A to 2B. 30. The MBM of specific example 29, wherein ABS3 includes the V _{H of the} anti-KLB antibody and / or V _L sequences, such as any of tables 2A to 2B, the pair of binder KLB V _H and / or V _L sequences. 31. Such as MBM in any one of specific examples 1 to 30, wherein ABS1 specifically binds to human KLB. 32. The MBM of specific example 31, wherein ABS1 includes the CDR sequence of the anti-KLB antibody, such as the CDR sequence of any KLB binder described in Tables 2A to 2B. 32 33. Specific examples of MBM, which comprises an anti-based ABS1 antibody -KLB V _H and / or V _L sequences, such as any of Tables 2A and 2B in the binder of a KLB V _H and / or V _L sequences . 34. The MBM of any one of specific examples 1 to 33, wherein ABS1 and ABS3 specifically bind to different epitopes on human KLB. 35. The MBM of specific example 34, wherein ABS1 and ABS3 can simultaneously specifically bind to their respective epitopes on human KLB. 36. The MBM of any one of Examples 1 to 35, wherein ABS2 specifically binds to the human fibroblast growth factor receptor isoform ("FGFR1c"). 37. The MBM of specific example 36, wherein ABS2 includes the CDR sequence of the anti-FGFR1c antibody, for example, the CDR sequence of any FGFR1c binder described in Tables 3A to 3B. 38. Specific examples of MBM 37, wherein the system comprises an anti-ABS2 antibody -FGFR1c V _H and / or V _L sequences, e.g. Table 3A-3B in any one of the binders of FGFR1c V _H and / or V _L sequences . 39. The MBM of any one of specific examples 36 to 38, wherein ABS2 is combined with the D3 loop of FGFR1c. 40. The MBM of any one of specific examples 36 to 83, wherein ABS2 is combined with the D2 loop of FGFR1c. 41. The MBM of any one of specific examples 1 to 27, wherein ABS2 specifically binds to human fibroblast growth factor receptor 3 ("FGFR3"). 42. The MBM of specific example 41, wherein ABS2 includes the CDR sequence of the anti-FGFR3 antibody, for example, the CDR sequence of any FGFR3 binder described in Table 4. 43. Specific examples of the MBM 42, wherein the system comprises an anti-ABS2 antibody -FGFR3 V _H and / or V _L sequences, e.g. in Table 4 according to any one of the FGFR3-binding subsequences of V _H and / or V _L sequence. 44. The MBM of any one of specific examples 1 to 27 or 41 to 43, wherein ABS3 specifically binds to human FGFR3. 45. The MBM of specific example 44, wherein ABS3 includes the CDR sequence of an anti-FGFR3 antibody, for example, the CDR sequence of any FGFR3 binder described in Table 4. 46. Specific examples of the MBM 45, wherein the system comprises an anti ABS3 antibody -FGFR3 V _H and / or V _L sequences, e.g. in Table 4 according to any one of the FGFR3-binding subsequences of V _H and / or V _L sequence. 47. The MBM of any one of specific examples 1 to 27, wherein ABS2 and ABS3 specifically bind to human FGFR3. 48. The MBM of Specific Example 47, wherein ABS2 includes the CDR sequence of the anti-FGFR3 antibody, for example, the CDR sequence of any FGFR3 binder described in Table 4. 49. Specific examples of the MBM 48, wherein the system comprises an anti-ABS2 antibody -FGFR3 V _H and / or V _L sequences, e.g. in Table 4 according to any one of the FGFR3-binding subsequences of V _H and / or V _L sequence. 50. The MBM of any one of specific examples 47 to 49, wherein ABS3 includes the CDR sequence of an anti-FGFR3 antibody, for example, the CDR sequence of any FGFR3 binder described in Table 4. 50 51. Specific examples of MBM, which comprises an anti-based ABS3 antibody -FGFR3 V _H and / or V _L sequences, e.g. in Table 4 according to any one of binder FGFR3 V _H and / or V _L sequence. 52. The MBM of any one of specific examples 47 to 51, wherein ABS2 and ABS3 specifically bind to the same epitope on human FGFR3. 53. Such as the MBM of specific example 52, wherein ABS2 and ABS3 include the same CDR sequence. 54. Such as the MBM of specific example 53, wherein ABS2 and ABS3 include the same V _H and V _L sequences. 55. The MBM of any one of specific examples 41 to 54, wherein ABS1 specifically binds to human CD63. 56. The MBM of specific example 55, wherein ABS1 includes the CDR sequence of the anti-CD63 antibody, for example, the CDR sequence of any CD63 binder described in Table 6. 56 57. Specific examples of MBM, which comprises ABS1 based antibody anti -CD63 V _H and / or V _L sequences, e.g., any of the binders CD63 V _H and / or V _L 6 in the Sequence Listing. 58. The MBM of any one of specific examples 41 to 54, wherein ABS1 specifically binds to human amyloid precursor protein 2 (APLP2). 59. The MBM of Specific Example 58, wherein ABS1 includes the CDR sequence of the anti-APLP2 antibody, for example, the CDR sequence of any APLP2 binder described in Table 5. 60. Specific examples of MBM 59, wherein the system comprises an anti-ABS1 antibody -APLP2 V _H and / or V _L sequences, e.g., any of the binders APLP2 V _H and / or V _L sequences described in Table 5. 61. A conjugate comprising MBM as in any one of specific examples 1 to 27 and a cytotoxic agent or cytostatic agent. 62. A pharmaceutical composition comprising MBM as in any one of Specific Examples 1 to 27 or a conjugate and excipient as in Specific Example 61. 63. A method of treating cancer, the method comprising administering to a subject suffering from cancer an effective amount of MBM as in any one of Specific Examples 1 to 27, such as the conjugate of Specific Example 61 or the medical composition as in Specific Example 62 Things. 64. The method of Specific Example 63, wherein the cancer is related to a target molecule that expresses and specifically binds to ABS1, ABS2, and/or ABS3. 65. The method of specific example 63 or specific example 64, wherein the MBM is an MBM according to any one of specific examples 41 to 60, and the conjugate includes the MBM according to any one of specific examples 41 to 60, or the medicine The composition system includes MBM according to any one of specific examples 41 to 60, or a binder including MBM according to any one of specific examples 41 to 60. 66. The method of any one of Specific Examples 63 to 65, wherein the cancer is bladder cancer. 67. The method of any one of Specific Examples 63 to 65, wherein the cancer is glioblastoma. 68. The method of any one of Specific Examples 63 to 65, wherein the cancer is squamous cell lung cancer. 69. A method for the treatment of non-alcoholic steatohepatitis ("NASH"), the method comprising administering to a subject suffering from NASH an effective amount of MBM as in any one of Specific Examples 1 to 27, as in Specific Example 61 Conjugate or medical composition as in Specific Example 62. 70. A method of treating a metabolic disease, the method comprising administering to a subject suffering from a metabolic disease an effective amount of MBM such as any one of specific examples 1 to 27, such as the conjugate of specific example 61 or such as specific examples 62 of the medical composition. 71. A method for lowering circulating DHL cholesterol, the method comprising administering to a subject suffering from elevated DHL cholesterol levels an effective amount of MBM such as any one of Examples 1 to 27, such as the conjugate of Example 61 or Such as the pharmaceutical composition of Specific Example 62. 72. A method for increasing circulating LDL cholesterol, the method comprising administering to a subject suffering from low LDL cholesterol an effective amount of MBM such as any one of Examples 1 to 27, such as the conjugate of Example 61 or such as Specific Example 62 of the pharmaceutical composition. 73. A method for lowering blood triglycerides, the method comprising administering to a subject suffering from elevated triglycerides an effective amount of MBM as in any one of Specific Examples 1 to 27, such as the combination of Specific Example 61 Or the pharmaceutical composition of Specific Example 62. 74. A method for lowering blood glucose, the method comprising administering to a subject suffering from elevated blood glucose an effective amount of MBM such as any one of specific examples 1 to 27, such as the conjugate of specific example 61 or such as specific example 62 The medical composition. 75. A method for treating obesity, the method comprising administering to a subject suffering from obesity an effective amount of MBM as in any one of specific examples 1 to 27, such as the conjugate of specific example 61 or as in specific example 62 Pharmaceutical composition. 76. A method for the treatment of diabetes, the method comprising administering to a subject suffering from diabetes an effective amount of MBM as in any one of specific examples 1 to 27, such as the conjugate of specific example 61 or the medical composition as in specific example 62 Things. 77. The method of any one of specific examples 69 to 76, wherein the MBM is an MBM according to any one of specific examples 28 to 40, and the conjugate includes the MBM according to any one of specific examples 28 to 40, or the medicine The composition system includes MBM according to any of Specific Examples 28 to 40, or a binder including MBM according to any of Specific Examples 28 to 40. 78. One or more nucleic acids encoding MBM as in any one of specific examples 1-60. 79. A cell that is engineered to behave as MBM in any of Specific Examples 1-60. 80. A cell transfected with one or more expression vectors, and the expression vector system includes one or more nucleic acid sequences encoding MBM as in any one of specific examples 1 to 60, the nucleic acid sequences being one or more Promoter control. 81. A method of manufacturing MBM, the method comprising: (a) culturing the cell of specific example 79 or 80 under conditions that express the MBM; and (b) recovering the MBM from the cell culture. 82. As the method of Specific Example 81, which further includes enriching the MBM. 83. The method of Specific Example 81 or Specific Example 82, which further includes purifying the MBM. 9. Citation of references

All publications, patents, patent applications and other documents cited in this application are incorporated herein by reference in their entirety for all purposes, and the degree of citation is as if each individual publication, patent or patent application or The other documents are individually and specifically incorporated by reference in their entirety for all purposes. In the case of inconsistencies between the teachings of one or more references incorporated in the text and this text, the teachings of this specification shall prevail.

(1): heavy chain variable region (2): linker (3): heavy chain variable region (4): linker (5): heavy chain variable region (6): CH1 constant domain (7): Optional hinge region (8): CH2 domain (9): CH3 domain (10): light chain variable region (11): constant domain (12): heavy chain variable region (13): constant domain ( 14): Hinge domain that exists as needed (15): CH2 domain (16): CH3 domain (17): light chain variable region (18): constant domain

Figure 1 : Schematic diagram of an exemplary MBM in this article.

Figures 2A to 2E : Representative images of the MBM in this article, where the Fab line contains modifications that promote proper VH and VL pairing. Additional strategies are stated in section 6.2.2 below.

Figures 3A to 3D : schematic diagrams of exemplary MBMs herein, where the Fc domain contains modifications to facilitate heterodimerization or purification of appropriately paired heterodimers. Figures 3A and 3B depict CH3 with a star-shaped mutation (CH3 with H435R Y436F modification) as CH3 (*) and Figure 3C and Figure 3D depict knobs denoted by (K) and (H), respectively. ) And hole mutations. Elsewhere in the figure, the stellar mutation system is depicted with an asterisk (*) and the club-and-mortar mutation system is depicted with a triangle (for example, ► or ◄). These two strategies (such as stellar mutation and club-and-hole mutation) can be combined in a single MBM. Additional heterodimerization strategies are described in section 6.2.4 below.

FIGS. 4A1 to 4B2: Exemplary expression pattern of a target molecule with specificity for the article of MBM: KLB and FGFR1c, according to genotype tissue performance (GTEx) program (FIG 4A1- FIG. 4A2) and human protein profile (Fig 4B1 To Figure 4B2). Organizations with overlapping manifestations are indicated in bold.

Figure 5 : Metabolic pathway regulated by FGF21 signaling.

Figures 6A to 6C : FACS binding analysis results (Example 1). Figure 6A: HEK293/Cas90hFGFR1/hKLB cells; Figure 6B: HEK293/hFGFR1c cells; Figure 6C: HEK293/hFGFR1c/hKLB cells. MFI: average fluorescence intensity; APC: isophycocyanin.

Figure 7 : Comparing to FGFR21, the bispecific binding molecule (BBM) REGN4366, FGFR1c x KLB bispecific control and the comparator FGFR1c x KLB bispecific REGN4304 are moderate in HEK293/SRE-luc/hFGFR1c/hKLB cells Schema of activation.

Figure 8 : Compared to the bivalent REGN4366, the trivalent F1K-scFv6 induced enhanced receptor activation (RLU: relative light unit) in HEK293.SREluc.hFGFR1cHS/hKLB cells (Example 1).

Figure 9 : Shows the evaluation of the linker length variants of 6 designated scFv6 molecules (contains the KLB binder named 22393 or 393 at position (1), and the FGFR1 binder named ADI-19842 or 842 at position (2) , There is a KLB binder named 22532 or 532 at position (3), which will not block the binding of KLB binder 22393 in scFv mode) the pattern of the results of the study. Compare molecules containing 7 to 45 amino acid linkers between the FGFR1 binding region and the N-terminal 532 scFv region. All three-specific binding molecules of linker length exhibited greater activity than the comparator bispecific molecule REGN4304.

Figures 10A to 10B : Schematic system showing that the three specific binders named F1K_scFv6 and scFv6 LK7 (linkers containing 7 amino acids) strongly activate FGFR1c signaling in HEK293 cells stably expressing hFGFR1c and hKLB. Figure 10A: Illustrates the western blot of the concentration-dependent FGFR1c signal delivered by ERK and PLCγ phosphorylation after serum starvation for 16 hrs followed by treatment with 1 nM and 10 nM drugs for 15 min. Figure 10B: Illustrates the Western ink delivered by the time-dependent FGFR1c signal of ERK and PLCγ phosphorylation after serum starvation for 16 hrs followed by drug treatment with a concentration of 10 nM for 15, 30 min and 1, 2, 4, and 6 hours of incubation time point.

Figures 11A to 11C : Compared with trispecific mAbs (F1K.scFv6 IgG1 and F1K-Fab6 IgG1), monospecific (anti-KLB; REGN4661) and bispecific binding molecules (anti-KLBxFGFR1c; REGN4304) are different The stoichiometric number is combined with KLB (solid circles in series)/FGFR1c (hollow rectangles). Figure 11A: Analysis of REGN4661: KLB complex (solid line) with asymmetric flow field flow combined with multi-angle light scattering (A4F-MALS). It also covers fractal maps from individual REGN4661 (dotted line) and KLB (dotted line) samples. Shows the relative UV absorbance at 215 nm of each sample with the residence time and indicates the measured molar mass of the analytical peak. Figure 11B: Analysis of REGN4304: KLB complex (thick solid line) and REGN4304: KLB: FGFR1c complex (thin solid line) with asymmetric flow field flow combined with multi-angle light scattering (A4F-MALS). It also covers fractal images from individual REGN4304 (dotted line), KLB (dotted dotted line) and FGFR1c (grey dotted dotted line) samples. Show the relative UV absorbance at 215 nm of each sample with the residence time and indicate the measured molar mass of the separated peak. Figure 11C: Analysis of F1K-scFv6 IgG1: KLB complex (thick solid line) and F1K-scFv6 IgG1: KLB: FGFR1c complex (0.2μM: 0.2) with asymmetric flow field flow combined with multi-angle light scattering (A4F-MALS) μM: 0.2 μM, thin solid line). Show the relative UV absorbance at 215 nm of each sample with the residence time and indicate the measured molar mass of the separated peak.

Figures 12A to 12B : How the clusters of FGFR1c, KLB, and FGF21 form active complexes (Figure 12A) and compared to bispecific molecules and monospecific controls, in FGFR1c and KLB receptors and 2+1 N-scFv three A schematic diagram of possible stoichiometric complexes formed between specific binding molecules, such as F1K-scFv6 (Figure 12B).

Figures 13A to 13B : Proliferation analysis of UMUC14 cells showing FGFR3 S249C mutation (Figure 13A) or FGFR3-TACC3 fusion mutation (Figure 13B).

Figure 14 Result of FGFR3 receptor degradation in UMUC14 cells.

(1): heavy chain variable region

(2): Linker

(3): heavy chain variable region

(4): Linker

(5): heavy chain variable region

(6): CH1 constant domain

(7): Hinge area that exists as needed

(8): CH2 domain

(9): CH3 domain

(10): Light chain variable region

(11): Constant domain

(12): heavy chain variable region

(13): Constant domain

(14): Hinge domain that exists as needed

(15): CH2 domain

(16): CH3 domain

(17): Light chain variable region

(18): Constant domain

Claims (89)

A multispecific binding molecule (MBM), which includes: (a) The first polypeptide chain, from N- to C-terminus, includes (i) scFv containing the first antigen binding site ("ABS1"), which is operably linked to (ii) the first Fab( "Fab1") the first heavy chain region, and the first Fab is operably linked to (iii) the Fc domain; (b) The second polypeptide chain, from N- to C-terminus, includes (i) the second heavy chain region of the second Fab ("Fab2"), and the second Fab is operably linked ( ii) Fc domain; (c) The third polypeptide chain, which includes the first light chain and the first heavy chain region paired to form Fab1, where Fab1 includes the second antigen binding site ("ABS2"); and (d) The fourth polypeptide chain, which includes the second light chain and the second heavy chain region paired to form Fab2, where Fab2 includes the third antigen binding site ("ABS3"). Such as the MBM of claim 1, wherein each antigen binding site ("ABS") binds to a different epitope. Such as the MBM of claim 1 or claim 2, wherein two of ABS1, ABS2 and ABS3 specifically bind to different epitopes of the same target molecule. The MBM of any one of claims 1 to 3, wherein the scFv, Fab1 and Fab2 can simultaneously specifically bind to their respective targets. Such as the MBM of any one of claims 1 to 4, wherein at least one of ABS1, ABS2, and ABS3 specifically binds to a target molecule having a first tissue performance profile, and at least one of ABS1, ABS2, and ABS3 is related to a second The target molecule of the tissue presentation profile specifically binds, and the second tissue presentation profile overlaps with the first tissue presentation profile, but is not the same. Such as the MBM of claim 5, wherein the first organization performance map and the second organization performance map overlap by 10 or less organizations. Such as the MBM of claim 6, wherein the first organization performance map and the second organization performance map overlap by 5 or less organizations. Such as the MBM of claim 7, wherein the first organization performance map and the second organization performance map overlap by 3 or less organizations. Such as the MBM of any one of claims 6 to 8, wherein the first and second tissue expression profiles are defined by the human protein map (HPA) and/or genotypic tissue expression (GTEx) project. Such as the MBM of any one of claims 6 to 9, wherein the first and second tissue expression profiles are protein expression profiles. Such as the MBM of any one of claims 6 to 9, wherein the first and second tissue performance profiles are mRNA performance profiles. For example, the MBM of any one of claims 1 to 11, wherein the affinity of the MBM to the target molecule of Fab1 is lower than the affinity of the second MBM to the target molecule of Fab1, and the second MBM lacks the scFv of the MBM but the rest is MBM is identical in amino acid sequence. Such as the MBM of any one of claims 1 to 12, wherein the scFv is connected to the first heavy chain region via a linker. Such as the MBM of request 13, where the linker: (a) The length is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids; and as needed (b) The length is at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids. Such as the MBM of request 14, where the linker: (a) The length is from 5 amino acids to 50 amino acids; (b) The length is from 5 amino acids to 45 amino acids; (c) The length is from 5 amino acids to 40 amino acids; (d) The length is from 5 amino acids to 35 amino acids; (e) The length is from 5 amino acids to 30 amino acids; (f) The length is from 5 amino acids to 25 amino acids; or (g) The length is from 5 amino acids to 20 amino acids. Such as the MBM of request 14, where the linker: (a) The length is from 6 amino acids to 50 amino acids; (b) The length is from 6 amino acids to 45 amino acids; (c) The length is from 6 amino acids to 40 amino acids; (d) The length is from 6 amino acids to 35 amino acids; (e) The length is from 6 amino acids to 30 amino acids; (f) The length is from 6 amino acids to 25 amino acids; or (g) The length is from 6 amino acids to 20 amino acids, Such as the MBM of request 14, where the linker: (a) The length is 7 to 40 amino acids; (b) The length is 7 amino acids to 35 amino acids; (c) The length is 7 to 30 amino acids; (d) The length is 7 amino acids to 25 amino acids; (e) The length is 7 to 20 amino acids. Such as the MBM of claim 14, wherein the length of the linker ranges from 5 amino acids to 25 amino acids. Such as the MBM of claim 14, wherein the linker has a length of 10 amino acids to 60 amino acids. Such as the MBM of claim 19, wherein the linker has a length of 20 amino acids to 50 amino acids. Such as the MBM of claim 20, wherein the linker has a length of 25 to 35 amino acids. Such as the MBM of any one of claims 13 to 21, wherein the linker is or includes a _{multimer of G n} S (SEQ ID NO: 61) or SG _n (SEQ ID NO: 62), wherein n is 1 to Integer of 7. Such as the MBM of claim 22, wherein the linker is or includes a _{multimer of G 4} S (SEQ ID NO: 4). Such as the MBM of any one of claims 13 to 23, wherein the linker is or includes two consecutive glycines (2Gly), three consecutive glycines (3Gly), and four consecutive glycines (4Gly (SEQ ID NO: 5)), five consecutive glycines (5Gly (SEQ ID NO: 6)), six consecutive glycines (6Gly (SEQ ID NO: 7)), seven consecutive glycines (7Gly ( SEQ ID NO: 8)), eight consecutive glycines (8Gly (SEQ ID NO: 9)) or nine consecutive glycines (9Gly (SEQ ID NO: 10)). The MBM according to any one of claims 1 to 24, wherein at least one of ABS1, ABS2, and ABS3 specifically binds to a membrane-bound antigen. Such as the MBM of claims 1 to 25, wherein ABS1 and ABS3 specifically bind to membrane-bound antigens on the same cell. Such as the MBM of any one of claims 1 to 26, which is a trispecific binding molecule ("TBM"). Such as the MBM of any one of claims 1 to 27, wherein the first light chain and the second light chain are universal light chains. Such as the MBM of any one of claims 1 to 27, wherein the light chain constant domain and the first heavy chain constant domain (CH1) of the first Fab or the second Fab are arranged in Crossmab. Such as the MBM of any one of claims 1 to 29, which includes an Fc heterodimer. Such as the MBM of claim 30, wherein the Fc domain in the Fc heterodimer, compared with the wild-type Fc domain, includes a knob-in-hole mutation. Such as the MBM of claim 30 or claim 31, wherein at least one Fc domain in the Fc heterodimer includes a star-shaped mutation compared to the wild-type Fc domain. Such as the MBM of any one of claims 1 to 32, it is a trivalent MBM. Such as the MBM of any one of claims 1 to 33, wherein ABS3 specifically binds to human klotho beta ("KLB"). Such as the MBM of claim 34, wherein ABS3 includes the CDR sequence of an anti-KLB antibody. Such as the MBM of claim 35, wherein ABS3 includes the V _H and/or V _L sequence of an anti-KLB antibody Such as the MBM of any one of claims 1 to 36, wherein ABS1 specifically binds to human KLB. Such as the MBM of claim 37, wherein ABS1 includes the CDR sequence of the anti-KLB antibody. The requested item of MBM 38, wherein the system comprises a V _H ABS1 -KLB anti-antibody, and / or V _L sequence. Such as the MBM of any one of claims 1 to 33, wherein ABS1 and ABS3 specifically bind to different epitopes on human KLB. Such as the MBM of claim 40, in which ABS1 and ABS3 can simultaneously specifically bind to their individual epitopes on human KLB. The MBM of any one of claims 1 to 41, wherein ABS2 specifically binds to the human fibroblast growth factor receptor 1c isoform ("FGFR1c"). Such as the MBM of claim 42, wherein ABS2 includes the CDR sequence of the anti-FGFR1c antibody. The requested item of MBM 43, wherein the system comprises a V _H ABS2 -FGFR1c anti-antibody, and / or V _L sequence. Such as the MBM of any one of claims 42 to 44, wherein ABS2 is combined with the D3 loop of FGFR1c. Such as the MBM of claims 42 to 44, in which ABS2 is combined with the D2 loop of FGFR1c. The MBM of any one of claims 1 to 33, wherein ABS2 specifically binds to human fibroblast growth factor receptor 3 ("FGFR3"). Such as the MBM of claim 47, wherein ABS2 includes the CDR sequence of an anti-FGFR3 antibody. The requested item of MBM 48, wherein the system comprises an anti-ABS2 antibody -FGFR3 V _H and / or V _L sequence. The MBM of any one of claims 1 to 33 or 47 to 49, wherein ABS3 specifically binds to human FGFR3. Such as the MBM of claim 50, wherein ABS3 includes the CDR sequence of an anti-FGFR3 antibody. The request of MBM item 51, wherein the system comprises an anti ABS3 antibody -FGFR3 V _H and / or V _L sequence. Such as the MBM of any one of claims 1 to 33, wherein ABS2 and ABS3 specifically bind to human FGFR3. Such as the MBM of claim 53, wherein ABS2 includes the CDR sequence of an anti-FGFR3 antibody. The requested item of MBM 54, wherein the system comprises an anti-ABS2 antibody -FGFR3 V _H and / or V _L sequence. The MBM of any one of claims 53 to 57, wherein ABS3 includes the CDR sequence of an anti-FGFR3 antibody. The requested item of MBM 56, wherein the system comprises an anti ABS3 antibody -FGFR3 V _H and / or V _L sequence. The MBM of any one of claims 53 to 57, wherein ABS2 and ABS3 specifically bind to the same epitope on human FGFR3. Such as the MBM of claim 58, where ABS2 and ABS3 include the same CDR sequence. Such as the MBM of claim 59, where ABS2 and ABS3 include the same V _H and V _L sequences. Such as the MBM of any one of claims 47 to 60, wherein ABS1 specifically binds to human CD63. Such as the MBM of claim 61, wherein ABS1 includes the CDR sequence of an anti-CD63 antibody. The requested item 62 of MBM, which comprises ABS1 based antibody anti -CD63 V _H and / or V _L sequence. Such as the MBM of any one of claims 47 to 60, wherein ABS1 specifically binds to human amyloid precursor-like protein 2 (APLP2). Such as the MBM of claim 64, wherein ABS1 includes the CDR sequence of the anti-APLP2 antibody. The request of MBM item 65, wherein the system comprises a V _H ABS1 -APLP2 an anti-antibody, and / or V _L sequence. A conjugate comprising MBM according to any one of claims 1 to 33 and a cytotoxic agent or cytostatic agent. A pharmaceutical composition comprising the MBM of any one of claims 1 to 33 or the conjugate and excipients of claim 67. A method for treating cancer, the method comprising administering to a subject suffering from cancer an effective amount of MBM such as any one of claims 1 to 33, a conjugate such as claim 67, or a medical composition such as claim 68 Things. The method of claim, wherein the cancer is related to the performance of the target molecule that specifically binds to ABS1, ABS2, and/or ABS3. Such as claim 69 or the method according to claim 70, wherein the MBM is an MBM according to any one of claims 47 to 66, and the conjugant includes an MBM according to any one of claims 47 to 66, or the medical composition The material system includes the MBM according to any one of claims 47 to 66, or a binder including the MBM according to any one of claims 47 to 66. The method according to any one of claims 69 to 71, wherein the cancer is bladder cancer. The method according to any one of claims 69 to 71, wherein the cancer is glioblastoma. The method according to any one of claims 69 to 71, wherein the cancer is squamous cell lung cancer. A method for the treatment of non-alcoholic steatohepatitis ("NASH"), the method comprising administering to a subject suffering from NASH an effective amount of MBM such as any one of claims 1 to 33, such as the combination of claim 67 , Or a pharmaceutical composition as in Claim 68. A method for treating metabolic diseases, the method comprising administering to a subject suffering from metabolic diseases an effective amount of MBM as in any one of claims 1 to 33, a conjugate as in claim 67, or as in claims 68 of the medical composition. A method of lowering circulating DHL cholesterol, the method comprising administering an effective amount of MBM such as any one of claims 1 to 33, such as the conjugate of claim 67, to a subject suffering from elevated DHL cholesterol levels, or Such as the medical composition of claim 68. A method for increasing circulating LDL cholesterol, the method comprising administering to a subject suffering from low LDL cholesterol an effective amount of MBM such as any one of claims 1 to 33, such as the conjugate of claim 67, or such as The medical composition of Claim 68. A method for lowering blood triglycerides, the method comprising administering an effective amount of the MBM of any one of claims 1 to 33 and the conjugate of claim 67 to a subject suffering from elevated triglyceride levels , Or the pharmaceutical composition of claim 68. A method for lowering blood glucose, the method comprising administering to a subject suffering from elevated blood glucose an effective amount of MBM such as any one of claims 1 to 33, a conjugate such as claim 67, or such as claim 68 of the medical composition. A method for treating obesity, the method comprising administering to a subject suffering from obesity an effective amount of MBM as in any one of claims 1 to 33, a conjugate as in claim 67, or as in claim 68 Pharmaceutical composition. A method for the treatment of diabetes, the method comprising administering to a subject suffering from diabetes an effective amount of MBM such as any one of claims 1 to 33, a conjugate such as claim 67, or a medical composition such as claim 68 Things. Such as the method according to any one of claims 75 to 82, wherein the MBM is an MBM according to any one of claims 34 to 46, and the joint includes the MBM according to any one of claims 34 to 46, or the The medical composition includes the MBM according to any one of claims 34 to 46, or a conjugate including the MBM according to any one of claims 34 to 46. One or more nucleic acids, which encode MBM according to any one of claims 1 to 66. A cell that is engineered to express MBM as in any one of claims 1 to 66. A cell transfected with one or more expression vectors, the expression vector system comprising one or more nucleic acid sequences encoding MBM according to any one of claims 1 to 66, the nucleic acid sequences being one or more promoters Under control. A method of manufacturing MBM, the method includes: (a) Under the condition of expressing the MBM, cultivate the cells of claim 85 or 86; and (b) Recover the MBM from the cell culture. Such as the method of claim 87, which further includes enriching the MBM. Such as the method of claim 87 or claim 88, which further includes purifying the MBM.

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